Fluorometric Detection of Active Alkaline Phosphatase and Gamma-Glutamyl Transferase in Fluid Dairy Products from Multiple Species

Ziobro, George C.; McELROY, Kevin M.

doi:10.4315/0362-028x.jfp-12-302

Cited by 8 publications

(7 citation statements)

References 24 publications

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“…To assay for ALP activity in breast milk, we modified a fluorometric detection method already published [35, 36]. Briefly, the samples were thawed, then vortexed to re-incorporate any separated cream, and diluted 1:10 in 100 mM Tris, pH 9.5.…”

Section: Methodsmentioning

confidence: 99%

Ontogeny of alkaline phosphatase activity in infant intestines and breast milk

et al. 2019

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BackgroundNecrotizing enterocolitis (NEC) is a devastating disease of intestinal inflammation that primarily affects premature infants. A potential risk factor for necrotizing enterocolitis is exposure of the premature neonatal intestine to environmental bacteria and their proinflammatory products such as lipopolysaccharide. The metalloenzyme alkaline phosphatase (ALP) has been shown to reduce lipopolysaccharide-mediated inflammation. Additionally, premature rat pups have reduced alkaline phosphatase activity and expression as compared to full term pups. To explore the possibility that the human premature neonatal intestine has a paucity of alkaline phosphatase activity, we measured endogenously produced intestinal alkaline phosphatase activity in meconium as a function of gestational age. To test whether breast milk could serve as a source of exogenous alkaline phosphatase to the neonatal intestine through ingestion, we measured alkaline phosphatase activity in breast milk across a range of time points post-birth.MethodsAlkaline phosphatase activity was quantified in 122 meconium samples from infants of gestational ages ranging from 24 to 40 weeks and in 289 breast milk samples collected from 78 individual mothers between days 2–49 post-birth.ResultsWe observed a strong positive correlation between the meconium alkaline phosphatase activity and gestational age, with preterm infants having lower meconium alkaline phosphatase activities than early term or term infants. Breast milk alkaline phosphatase activity was highest in the first week post-birth, with peak alkaline phosphatase activity at day 2 post-birth, followed by relatively low alkaline phosphatase activity in weeks 2–7.ConclusionsOur results are consistent with the two major risk factors for necrotizing enterocolitis development, preterm birth and lack of breast milk feeding, both contributing to a paucity of alkaline phosphatase activity and impaired capacity to detoxify proinflammatory bacterial products such as lipopolysaccharide.

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Section: Methodsmentioning

confidence: 99%

Ontogeny of alkaline phosphatase activity in infant intestines and breast milk

et al. 2019

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“…A fluorescence method based on the dephosphorylation of 4‐methyl‐umbelliferone‐phosphate was first published by Fernley and Walker (1965) for the analysis of purified ALP activity. A method with the same substrate was later adapted for liquid dairy products by Ziobro and McElroy (2013) and has now been further improved for the analysis of liquid and solid dairy products and will be published in the near future. This microplate method will also be published within ISO as a technical specification 17.…”

Section: Assessmentmentioning

confidence: 99%

“…Synthetic substrates are available to monitor the activity of GGT, either by spectrophotometric analysis (g‐glutamyl‐p‐nitroanilide) (Zehetner et al., 1995) or by fluorescence measurement (L‐glutamic acid‐y‐7‐Amino‐4‐methylcoumarin) (Ziobro and McElroy, 2013).…”

Section: Assessmentmentioning

confidence: 99%

“…The heat inactivation curve of this enzyme occurs in cows' milk between 65°C and 76°C (Zehetner et al, 1995) and therefore, the inactivation properties can be used to monitor milk pasteurisation (Zehetner et al, 1995;Ziobro and McElroy, 2013). Vetsina et al (2014) considered GGT as a useful marker for bovine milk pasteurisation.…”

Section: C-glutamyl Transferase (Ggt)mentioning

confidence: 99%

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The use of alkaline phosphatase and possible alternative testing to verify pasteurisation of raw milk, colostrum, dairy and colostrum‐based products

Clawin‐Rädecker¹,

Block²,

Egger³

et al. 2021

EFS2

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Pasteurisation of raw milk, colostrum, dairy or colostrum-based products must be achieved using at least 72°C for 15 s, at least 63°C for 30 min or any equivalent combination, such that the alkaline phosphatase (ALP) test immediately after such treatment gives a negative result. For cows' milk, a negative result is when the measured activity is ≤ 350 milliunits of enzyme activity per litre (mU/L) using the ISO standard 11816-1. The use and limitations of an ALP test and possible alternative methods for verifying pasteurisation of those products from other animal species (in particular sheep and goats) were evaluated. The current limitations of ALP testing of bovine products also apply. ALP activity in raw ovine milk appears to be about three times higher and in caprine milk about five times lower than in bovine milk and is highly variable between breeds. It is influenced by season, lactation stage and fat content. Assuming a similar pathogen inactivation rate to cows' milk and based on the available data, there is 95-99% probability (extremely likely) that pasteurised goat milk and pasteurised sheep milk would have an ALP activity below a limit of 300 and 500 mU/L, respectively. The main alternative methods currently used are temperature monitoring using data loggers (which cannot detect other process failures such as cracked or leaking plates) and the enumeration of Enterobacteriaceae (which is not suitable for pasteurisation verification but is relevant for hygiene monitoring). The inactivation of certain enzymes other than ALP may be more suitable for the verification of pasteurisation but requires further study. Secondary products of heat treatment are not suitable as pasteurisation markers due to the high temperatures needed for their production. More research is needed to facilitate a definitive conclusion on the applicability of changes in native whey proteins as pasteurisation markers.

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“…Alkaline phosphatase activity has been used to confirm the efficacy of pasteurization in dairy products. Thus, a dairy product that contains an insignificant amount of active enzyme or no enzyme at all is considered properly pasteurized [9].…”

Section: Introductionmentioning

confidence: 99%

Correlation Analysis of Microbial Contamination and Alkaline Phosphatase Activity in Raw Milk and Dairy Products

Zixin

Liu

Yang

et al. 2023

IJERPH

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Microbial contamination in raw milk and dairy products can detrimentally affect product quality and human health. In this study, the aerobic plate count, aerobic Bacillus abundance, thermophilic aerobic Bacillus abundance, and alkaline phosphatase activity were determined in 435 raw milk, 451 pasteurized milk, and 617 sterilized milk samples collected from 13 Chinese provinces (or municipalities). Approximately 9.89% and 2.22% of raw milk and pasteurized milk samples exceeded the threshold values for the aerobic plate count, respectively. The proportions of aerobic Bacillus in raw milk, pasteurized milk, and sterilized milk were 54.02%, 14.41%, and 1.30%, respectively. The proportions of thermophilic aerobic Bacillus species were 7.36% in raw milk and 4.88% in pasteurized milk samples, and no bacteria were counted in sterilized milk. Approximately 36.18% of raw milk samples contained >500,000 mU/L of alkaline phosphatase activity, while 9.71% of pasteurized milk samples contained >350 mU/L. For raw milk, there was a positive correlation between the aerobic plate count, the aerobic Bacillus abundance, and the alkaline phosphatase activity, and there was a positive correlation between the aerobic Bacillus abundance, the thermophilic aerobic Bacillus count, and the alkaline phosphatase activity. For pasteurized milk, there was a positive correlation between the aerobic plate count, the aerobic Bacillus abundance, and the thermophilic aerobic Bacillus count; however, the alkaline phosphatase activity had a negative correlation with the aerobic plate count, the aerobic Bacillus abundance, and the thermophilic aerobic Bacillus abundance. These results facilitate the awareness of public health safety issues and the involvement of dairy product regulatory agencies in China.

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Fluorometric Detection of Active Alkaline Phosphatase and Gamma-Glutamyl Transferase in Fluid Dairy Products from Multiple Species

Cited by 8 publications

References 24 publications

Ontogeny of alkaline phosphatase activity in infant intestines and breast milk

Ontogeny of alkaline phosphatase activity in infant intestines and breast milk

The use of alkaline phosphatase and possible alternative testing to verify pasteurisation of raw milk, colostrum, dairy and colostrum‐based products

Correlation Analysis of Microbial Contamination and Alkaline Phosphatase Activity in Raw Milk and Dairy Products

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