Raw camel milk has gained significant popularity as a functional and nutraceutical food beyond traditional arid regions due to its unique bioactive compounds and health benefits
(Tidjani et al., 2025). This trend reflects increasing health consciousness among consumers and has expanded into new applications, including infant formulas for those with cow milk allergies
(Ho et al., 2022).
Microbiological safety concerns arise from raw camel milk’s diverse microbial flora, which contains both beneficial microorganisms and potential pathogens. These concerns are heightened by improper handling and poor hygiene, especially in developing countries where foodborne illnesses significantly impact public health (
Tomar and Tiwari, 2024).
As global consumption increases, processing methods like pasteurization, microwave treatment and controlled fermentation are essential for ensuring safety and extending shelf life
(Lankri et al., 2024). Standard microbiological assessment typically employs methods for enumerating re
viable mesophilic aerobic microflora (RAMF), such as total
viable count (TVC) or aerobic plate count (APC), with plate count agar (PCA) serving as the primary culture medium
(Drici et al., 2023).
However, PCA may not effectively recover the complete range of cultivable microbes in raw camel milk, potentially underestimating microbial populations. Addressing this limitation, researchers have developed camel milk-plate count agar (CaM-PCA), an innovative medium designed to enhance accuracy, sensitivity and efficiency in RAMF enumeration and detection of specific bacterial species in raw camel milk.
This specialized culture medium offers an effective solution for improving microbiological assessment and enhancing safety control of raw camel milk as it continues to gain popularity as a nutraceutical dairy product with exceptional health properties.
Raw camel milk sampling and preparation
Twenty-three raw camel milk samples were collected from food stores (n=15) and farms (n=8) in Southern Algeria in February-March 2022. This small sample size reflects challenges obtaining raw camel milk in the region, where production is seasonal, limited and mostly consumed locally. Samples underwent immediate on-site California mastitis test and pH measurement before transport in cool boxes (6
oC) within 20 minutes, following standard protocols to maintain sample integrity (
Kroger, 1985). Upon arrival at the Sciences and Environment research laboratory (SCIENV-C1810200, University of Tamanghasset), samples were subjected to pH remeasurement and microbiological analysis for RAMF enumeration to assess microbial load.
Sterilized milk-based culture medium CaM-PCA com- position and preparation
Raw camel milk samples were tested for stability under microwave or autoclave sterilization at original pH or when adjusted to pH 7.1-7.2 with 0.1 M NaOH (
Davies and White, 1966) The novel CaM-PCA medium was developed based on milk plate count agar (APHA and ISO 4833 standards), replacing skimmed milk with 1% (v/v) heat-stable camel milk (SP1). Composition included 5.00 g peptone water, 1.00 g glucose, 2.50 g yeast extract, 10 mL whole camel milk and 10 g agar per liter of distilled water. After boiling and complete dissolution, the medium was bottled, autoclaved (115
oC or 121
oC for 15 minutes), cooled to 50
oC and poured into plates (14 mL/dish). Plates were dried inverted for 72 hours at 24
oC before use.
RAMF spread plating and SP SDS culture methods
Standard surface spreading (
Johns and Mcnabb, 1930) and SP-SDS
(Thomas et al., 2015) methods were used to ensure that the RAMF enumeration was performed in duplicate through predried PCA and CaM-PCA Petri dishes. Tenfold serial dilution were performed from 10-1 to 10-6 for all raw camel milk samples and then 100 µL or 10 µL of each sample or serial dilution was plated or spotted on both PCA and CaM-PCA culture media. The readings were taken after 48 hours of incubation at 30
oC and the results are expressed in colony-forming units/mL (CFU/mL). CFU/mL counts were obtained from plates and spots as duplicates of appropriate dilutions of each raw camel milk sample.
Data analysis
Statistical analysis of the RAMF count was performed with trial version 2024.2.0 of XLSTAT Premium. The RAMF data were converted to base-10 logarithms of CFU/mL of the raw camel milk samples (log10 CFU/mL). Then, accounting for the small sample size (twenty three samples), the data were tested for the normality parameter using the Shapiro Wilk test. Finally, because the data did not follow the normal distribution rules, a nonparametric Mann Whitney test at a significance level of 5% was applied to compare the two studied groups, with PCA and CaM-PCA culture media used as independent variables and the microbiological count used as the dependent variable.
Stability of raw camel milk after sterilization
The twenty-three raw camel milk samples collected, all four samples from SP1 maintained their fluid structure and homogeneous appearance after autoclaving (115
oC or 121
oC for 15 minutes) or microwave treatment, regardless of pH adjustment (Table 1). In contrast, the remaining nineteen samples (83%) lost structure in sterilization step, showing coagulation and whey separation. One sample from SP4 exhibited browning after heat treatment, indicating Maillard reaction occurrence. The poor heat stability observed in most samples aligns with previous research on camel milk proteins (
Farah and Atkins, 1992;
Alhaj et al., 2011; Ho et al., 2022; Zhang et al., 2023) . This instability stems from the absence of β-lactoglobulin and reduced κ-casein levels (only 5% of total casein versus 13.6% in bovine milk), leading to protein coagulation within 2-3 minutes at 120
oC (
Farah and Atkins, 1992). The browning observed in SP4 samples likely resulted from Maillard reactions between lactose, casein lysine and whey proteins
(Mohamed et al., 2022; Zhao et al., 2023). The remarkable stability of SP1 samples (17%) represents a novel finding in camel milk heat treatment. This stability may be attributed to optimal κ-casein concentration, as adding 1-2 mg/mL κ-casein increases heat stability at pH 6.7-6.9
(Kappeler et al., 1998; Alhaj et al., 2011). Other factors may include protein compositions similar to cow milk, low lactose content, or the presence of heat stabilizers like phosphates or citrates
(Mohamed et al., 2022; Zhao et al., 2023).
RAMF enumeration on PCA versus CaM-PCA culture media
All twenty-three raw camel milk samples showed higher colony counts on CaM-PCA than on PCA medium at the same dilution (Table 2). Nonparametric Mann-Whitney test revealed significantly higher RAMF counts (p=0.048, α=0.05) on CaM-PCA (8.50 log10 CFU/mL ± 0.45) compared to PCA (8.20 log10 CFU/mL ± 0.60). Box plot analysis confirmed higher values with lower variation on CaM-PCA (SD=0.446) versus PCA (SD=0.595) (Fig 1). The CaM-PCA medium showed superior performance in enumerating microorganisms from raw camel milk, likely due to providing essential nutrients and growth factors specific to the camel milk ecosystem that are absent in conventional PCA. This parallels the development of milk plate count agar with 0.1% skimmed milk for dairy product analysis (
Wehr and Frank, 2004).
Molecular identification of CaM-PCA specific isolates
Three bacterial isolates (CaM-T9, CaM-T13 and CaM-T20) were uniquely detected on CaM-PCA medium. Based on 16S rRNA sequence similarity using the EzBioCloud database, CaM-T13 (PQ260741) was assigned to
Hafnia alvei (99.54% similarity), CaM-T20 (PQ260742) to
Enterobacter hormaechi subsp.
hoffmannii (99.65% similarity) and CaM-T9 (PQ260744) to the
Metabacillus genus (98.84% similarity with
M. halosaccharovorans and
M. schmidteae) (Fig 2). The unique detection of
Metabacillus in raw camel milk using CaM-PCA highlights its enhanced cultivation capabilities. Species of
Metabacillus genus are typically found in extreme environments, likely entered the milk through environmental contamination. Their spore-forming nature raises food safety concerns as these spores can resist pasteurization (
Patel and Gupta, 2020). Similarly, the opportunistic pathogens
Hafnia alvei and
Enterobacter hormaechi subsp.
hoffmannii indicates potential contamination through environmental sources, animal contact, human handlers, or dairy equipment biofilms. The psychrotrophic nature of
Hafnia alvei poses particular concerns for milk quality in cold storage
(Tabla et al., 2016).
