Impact of dietary regime on the metabolomic profile of bovine buttermilk and whole milk powder.

INTRODUCTION: Bovine milk contains a rich matrix of nutrients such as carbohydrates, fat, protein and various vitamins and minerals, the composition of which is altered by factors including dietary regime. OBJECTIVES: The objective of this research was to investigate the impact of dietary regime on the metabolite composition of bovine whole milk powder and buttermilk. METHODS: Bovine whole milk powder and buttermilk samples were obtained from spring-calving cows, consuming one of three diets. Group 1 grazed outdoors on perennial ryegrass which was supplemented with 5% concentrates; group 2 were maintained indoors and consumed a total mixed ration diet; and group 3 consumed a partial mixed ration diet consisting of perennial ryegrass during the day and total mixed ration maintained indoors at night. RESULTS: Metabolomic analysis of the whole milk powder (N = 27) and buttermilk (N = 29) samples was preformed using liquid chromatography-tandem mass spectrometry, with 504 and 134 metabolites identified in the samples respectively. In whole milk powder samples, a total of 174 metabolites from various compound classes were significantly different across dietary regimes (FDR adjusted p-value ≤ 0.05), including triglycerides, of which 66% had their highest levels in pasture-fed samples. Triglycerides with highest levels in pasture-fed samples were predominantly polyunsaturated with high total carbon number. Regarding buttermilk samples, metabolites significantly different across dietary regimes included phospholipids, sphingomyelins and an acylcarnitine. CONCLUSION: In conclusion the results reveal a significant impact of a pasture-fed dietary regime on the metabolite composition of bovine dairy products, with a particular impact on lipid compound classes.

Facial synthesis of fluorine-engineered magnetic covalent organic framework for selective and ultrasensitive determination of fipronil, its metabolites and analogs in food samples.

To improve the adsorption affinity and selectivity of fipronils (FPNs), including fipronil, its metabolites and analogs, a magnetic covalent organic framework (Fe3O4@COF-F) with copious fluorine affinity sites was innovatively designed as an adsorbent of magnetic solid-phase extraction (MSPE). The enhanced surface area, pore size, crystallinity of Fe3O4@COF-F and its exponential adsorption capacities (187.3-231.5 mg g-1) towards fipronils were investigated. Combining MSPE with high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS), an analytical method was established for the selective determination of fipronils in milk and milk powder samples. This method achieved high sensitivity (LODs: 0.004-0.075 ng g-1), satisfactory repeatability and accuracy with spiked recoveries ranging from 89.9% to 100.3% (RSDs≤5.1%). Overall, the constructed Fe3O4@COF-F displayed great potential for the selective enrichment of fipronils, which could be ascribed to fluorine­fluorine interaction. This method proposed a feasible and promising strategy for the development of functionalized COF and broadened its application in fluorine containing hazards detection.

Deep eutectic solvent-functionalized amorphous UiO-66 for efficient extraction and ultrasensitive analysis of perfluoroalkyl substances in infant milk powder.

In this study, a convenient and effective method for determination of perfluoroalkyl substances (PFASs) in infant formula was developed based on a novel dispersive solid-phase extraction using deep eutectic solvent-functionalized amorphous UiO-66 (DES/aUiO-66) as sorbent. The synthesis of materials could be achieved without the use of complex and environmentally unfriendly procedures. Parameters were systematically investigated to establish a simple, fast, and efficient green pretreatment method. The method demonstrated high sensitivity, good precision, a detection limit of 0.330-0.529 ng·kg-1, and low matrix effects (< 12.8%). The mechanism for this material was elucidated by ab initio molecular dynamics (AIMD) simulations and quantum chemistry calculations. The presence of massive pore structures and collectively synergistic binding sites facilitated affinity adsorption toward PFASs. Finally, this method was applied to the monitoring of PFASs in 10 actual milk powder samples. This groundbreaking approach opens new possibilities for the advancement of analytical techniques and food safety monitoring.

Diet Supplemented with Special Formula Milk Powder Promotes the Growth of the Brain in Rats.

This investigation was to study the effects of different formula components on the brain growth of rats. Fifty male SD rats were randomly divided into five groups: a basic diet group; a 20% ordinary milk powder group; a 20% special milk powder group; a 30% ordinary milk powder group; and a 30% special milk powder group by weight. LC-MS was used to detect brain lipidomics. After 28 days of feeding, compared with the basic diet group, the brain/body weights of rats in the 30% ordinary milk powder group were increased. The serum levels of 5-HIAA in the 30% ordinary milk powder group were lower than in the 20% ordinary milk powder group. Compared with the basic diet group, the expressions of DLCL, MePC, PI, and GM1 were higher in the groups with added special milk powder, while the expressions of LPE, LdMePE, SM, and MGTG were higher in the groups with added ordinary milk powder. The expression of MBP was significantly higher in the 20% ordinary group. This study found that different formula components of infant milk powder could affect brain growth in SD rats. The addition of special formula infant milk powder may have beneficial effects on rat brains by regulating brain lipid expression.

Evaluating the effectiveness of minimum chlorate technologies employed by manufacturers of skim milk powder from both microbiological quality and chemical residue perspectives.

Dairy processors in the Republic of Ireland have adopted chlorine-free chemicals for cleaning and chlorine gas for water disinfection as a means of minimizing chlorate residue in dairy products. For these 'minimum chlorate technologies' to be satisfactory, they must be able to deliver product with acceptable levels of bacteria as well as minimum levels of chlorate and other chlorine based residues. To establish the effectiveness of these technologies, sampling was conducted across the skim milk powder (SMP) manufacturing chain in 3 separate milk processing sites. Across the 3 sites a total of 11 different batches of SMP were sampled in duplicate from the whole milk silo through the manufacturing process to the powder product; yielding a total of 137 samples. Samples were tested for chlorate, perchlorate and trichloromethane alongside a suite of microbiological plate count tests including total bacteria, thermophilic bacteria, thermoduric bacteria and both mesophilic and thermophilic spore-forming bacteria. Chlorate was detected at reportable levels (≥0.01 mg/kg) in 9 of 22 SMP samples analyzed; resulting in a mean chlorate concentration 0.0183 mg/kg. Bacteria were ubiquitous across all samples analyzed with spore-forming bacteria counts ranging from 1.30 to 2.33 log cfu/ g in SMP.

Quality characteristics of goat milk powder produced by freeze drying followed by UV-C radiation sterilization.

Goat milk was directly freeze-dried into milk powder after freezing and then sterilized using UV-C radiation to produce low-dose, medium-dose and high-dose UV-C radiation sterilized freeze-dried goat milk powder (LGP, MGP and HGP). UV-C sterilization effectively reduced the total bacteria count and coliform bacteria in the goat milk powder while preserving the active proteins, and maintaining the color unchanged. Additionally, LGP, MGP, and HGP all exhibited a moisture content below 5 g/100 g and water activity below 0.5. Upon reconstitution, the milk powder formed uniform and stable emulsion. During accelerated storage tests, the increased Aw did not compromise the microbial quality of milk powder, and there were no significant changes in active proteins as confirmed via SDS-PAGE results. Furthermore, the color parameters (a*, b* and ΔE) showed a strong correlation with hydroxymethyl furfural levels.

A Dual and Rapid RPA-CRISPR/Cas12a Method for Simultaneous Detection of Cattle and Soybean-Derived Adulteration in Goat Milk Powder.

The adulteration of goat milk powder occurs frequently; cattle-derived and soybean-derived ingredients are common adulterants in goat milk powder. However, simultaneously and rapidly detecting cattle-derived and soybean-derived components is still a challenge. An efficient, high-throughput screening method for adulteration detection is needed. In this study, a rapid method was developed to detect the adulteration of common cattle-derived and soybean-derived components simultaneously in goat milk powder by combining the CRISPR/Cas12a system with recombinant polymerase amplification (RPA). A dual DNA extraction method was employed. Primers and crRNA for dual detection were designed and screened, and a series of condition optimizations were carried out in this experiment. The optimized assay rapidly detected cattle-derived and soybean-derived components in 40 min. The detection limits of both cattle-derived and soybean-derived components were 1% (w/w) for the mixed adulteration models. The established method was applied to a blind survey of 55 commercially available goat milk powder products. The results revealed that 36.36% of the samples contained cattle-derived or soybean-derived ingredients, which revealed the noticeable adulteration situation in the goat milk powder market. This study realized a fast flow of dual extraction, dual amplification, and dual detection of cattle-derived and soybean-derived components in goat milk powder for the first time. The method developed can be used for high-throughput and high-efficiency on-site primary screening of goat milk powder adulterants, and provides a technical reference for combating food adulteration.

Rapid and accurate quantification of viable Bifidobacterium cells in milk powder with a propidium monoazide - antibiotic fluorescence in situ hybridization - flow cytometry method.

Due to its beneficial effects on human health, Bifidobacterium is commonly added to milk powder. Accurate quantification of viable Bifidobacterium is essential for assessing the therapeutic efficacy of milk powder. In this study, we introduced a novel propidium monoazide (PMA) - antibiotic fluorescence in situ hybridization (AFISH) - flow cytometry (FCM) method to rapidly and accurately quantify viable Bifidobacterium cells in milk powder. Briefly, Bifidobacterium cells were treated with chloramphenicol (CM) to increase their rRNA content, followed by staining with RNA-binding oligonucleotide probes, based on the AFISH technique. Then, the DNA-binding dye PMA was used to differentiate between viable and non-viable cells. The PMA-AFISH-FCM method, including sample pretreatment, CM treatment, dual staining, and FCM analysis, required around 2 h and was found to be better than the current methods. This is the first study to implement FCM combined with PMA and oligonucleotide probe for detecting Bifidobacterium.

Mechanistic insights into the changes of biological activity and physicochemical characteristics in Lacticaseibacillus paracasei fortified milk powder during storage.

To clarify the change mechanism of biological activity and physicochemical characteristics in Lacticaseibacillus paracasei JY025 fortified milk powder (LFMP) during storage, morphological observation, JY025 survival, storage stability, and metabolomics of LFMP were determined during the storage period in this study. The results showed that the LFMP had a higher survival rate of JY025 compared with the bacterial powder of JY025 (LBP) during storage, which suggested that milk powder matrix could reduce strain JY025 mortality under prolonged storage in the LFMP samples. The fortification of strain JY025 also affected the stability of milk powder during the storage period. There was lower water activity and higher glass transition temperature in LFMP samples compared with blank control milk powder (BCMP) during storage. Moreover, the metabolomics results of LFMP indicated that vitamin degradation, Maillard reaction, lipid oxidation, tricarboxylic acid cycle, and lactobacilli metabolism are interrelated and influence each other to create complicated metabolism networks.

Reduction of maillard browning in spray dried low-lactose milk powders due to protein polysaccharide interactions.

Lactose hydrolysed concentrated milk was prepared using ß-galactosidase enzyme (4.76U/mL) with a reaction period of 12 h at 4 °C. Addition of polysaccharides (5 % maltodextrin/ß-cyclodextrin) to concentrated milk either before or after lactose hydrolysis did not result in significant differences (p > 0.05) in degree of hydrolysis (% DH) of lactose and residual lactose content (%). Three different inlet temperatures (165 °C, 175 °C and 185 °C) were used for the preparation of powders which were later characterised based on physico-chemical and maillard browning characteristics. Moisture content, solubility and available lysine content of the powders decreased significantly, whereas, browning parameters i.e., browning index, 5-hydroxymethylfurfural, furosine content increased significantly (p < 0.05) with an increase in inlet air temperature. The powder was finally prepared with 5 % polysaccharide and an inlet air temperature of 185 °C which reduced maillard browning. Protein-polysaccharide interactions were identified using Fourier Transform infrared spectroscopy, fluorescence spectroscopy and determination of free amino groups in the powder samples. Maltodextrin and ß-cyclodextrin containing powder samples exhibited lower free amino groups and higher degree of graft value as compared to control sample which indicated protein-polysaccharide interactions. Results obtained from Fourier Transform infrared spectroscopy also confirmed strong protein-polysaccharide interactions, moreover a significant decrease in fluorescence intensity was also observed in the powder samples. These interactions between the proteins and polysaccharides reduced the maillard browning in powders.

Dynamic Gastrointestinal Digestion of Bovine, Caprine and Ovine Milk Reconstituted from Commercial Whole Milk Powders.

The global dairy market has been increasingly diversified with more dairy product offerings of milk products from different animal species. Meanwhile, milk powders remain the main exported dairy product format due to their ease of transportation. In this work, we studied the structural changes, protein hydrolysis and nutrient delivery during dynamic gastric digestion and small intestinal digestion of cow, goat and sheep milk reconstituted from commercial whole milk powders. The results show that the reconstituted milks digest similarly to processed fresh milk. The digestion behaviors of the three reconstituted ruminant milks are broadly similar (gastric coagulation, kinetics of gastric emptying of protein and fat and the high digestibility in the small intestine) with some differences, which are likely contributed by the processing history of the milk powders. The delivery of individual amino acids to the small intestine differed between the early and late stages of gastric digestion, which were primarily affected by the abundance of amino acids in caseins and whey proteins but also by the difference between milk types associated with their gastric coagulation behaviors. This work showed that powdered milk is similar to fresh processed milk in digestion behavior, and the inherent differences between ruminant milks can be modified by processing treatments.

The Fast Quantification of Vitamin B12 in Milk Powder by High-Performance Liquid Chromatography-Inductively Coupled Plasma Mass Spectrometry.

This study presents a new technique for determining vitamin B12 in milk powder using high-performance liquid chromatography-inductively coupled plasma mass spectrometry (HPLC-ICP-MS). We used ultrasonics with potassium ferrocyanide and zinc acetate solutions to extract the samples. 59Co was employed as the analytical target for cyanocobalamin. It was separated using a Phenomenex Luna 5 µm C18 (250 × 4.6 mm) chromatographic column with a mobile phase consisting of 1.6 mmol/L EDTA and 0.4 mmol/L KH2PO4 in a 60% v/v methanol solution (pH = 4.0). The sample has an excellent separating degree for free cobalt and cyanocobalamin, and isocratic elution can be finished within 4.0 min. To eliminate the matrix interference due to the presence of milk powder, we applied collision mode (KED). The linear range of cyanocobalamine ranged from 1.0 µg/L to 20 µg/L, with correlation coefficients (r2) of 0.9994. The limit of detection (LOD) was 0.63 µg/kg, and the limit of quantitation (LOQ) was 2.11 µg/kg. The mean recoveries were in the range of 87.4-103.6%. The accuracy and precision of the developed method are well suited for the fast quantification of the trace vitamin B12 in milk powder.

Conformation-Activity Mechanism of Alcalase Hydrolysis for Reducing In Vitro Allergenicity of Instant Soy Milk Powder.

Effective reduction of the allergenicity of instant soy milk powder (ISMP) is practically valuable for expanding its applications. This study optimized the enzymolysis technology of ISMP using single-factor experiments and response surface methodology, combined serological analysis, cellular immunological models, bioinformatics tools, and multiple spectroscopy techniques to investigate the effects of alcalase hydrolysis on allergenicity, spatial conformation, and linear epitopes of ISMP. Under the optimal process, special IgE and IgG1 binding abilities and allergenic activity to induce cell degranulation of alcalase-hydrolyzed ISMP were reduced by (64.72 ± 1.76)%, (56.79 ± 3.72)%, and (73.3 ± 1.19)%, respectively (P < 0.05). Moreover, the spatial conformation of instant soy milk powder hydrolysates (ISMPH) changed, including decreased surface hydrophobicity, a weaker peak of amide II band, lower contents of α-helix and ß-sheet, and an enhanced content of random coil. Furthermore, the linear epitopes of major soy allergens, 9 from glycinin and 13 from ß-conglycinin, could be directionally disrupted by alcalase hydrolysis. Overall, the structure-activity mechanism of alcalase hydrolysis to reduce ISMP allergenicity in vitro was preliminarily clarified. It provided a new research direction for the breakthrough in the desensitization of ISMP and a theoretical basis for revealing the potential mechanism of alcalase enzymolysis to reduce the allergenicity of ISMP.

Bootstrapping for Estimating the Conservative Kill Ratio of the Surrogate to the Pathogen for Use in Thermal Process Validation at the Industrial Scale.

A surrogate is commonly used for process validations. The industry often uses the target log cycle reduction for the test (LCRTest) microorganism (surrogate) to be equal to the desired log cycle reduction for the target (LCRTarget) microorganism (pathogen). When the surrogate is too conservative with far greater resistance than the pathogen, the food may be overprocessed with quality and cost consequences. In aseptic processing, the Institute for Thermal Processing Specialists recommends using relative resistance (DTarget)/(DTest) to calculate LCRTest (product of LCRTarget and relative resistance). This method uses the mean values of DTarget and DTest and does not consider the estimating variability. We defined kill ratio (KR) as the inverse of relative resistance.The industry uses an extremely conservative KR of 1 in the validation of food processes for low-moisture foods, which ensures an adequate reduction of LCRTest, but can result in quality degradation. This study suggests an approach based on bootstrap sampling to determine conservative KR, leading to practical recommendations considering experimental and biological variability in food matrices. Previously collected thermal inactivation kinetics data of Salmonella spp. (target organism) and Enterococcus faecium (test organism) in Non-Fat Dried Milk (NFDM) and Whole Milk Powder (WMP) at 85, 90, and 95°C were used to calculate the mean KR. Bootstrapping was performed on mean inactivation rates to get a distribution of 1000 bootstrap KR values for each of the treatments. Based on minimum temperatures used in the industrial process and acceptable level of risk (e.g., 1, 5, or 10% of samples that would not achieve LCRTest), a conservative KR value can be estimated. Consistently, KR increased with temperature and KR for WMP was higher than NFDM. Food industries may use this framework based on the minimum processing temperature and acceptable level of risk for process validations to minimize quality degradation.

A Magnetic Beads-Based Sandwich Chemiluminescence Enzyme Immunoassay for the Rapid and Automatic Detection of Lactoferrin in Milk.

Lactoferrin (LF), an iron-binding glycoprotein with immunological properties and a high nutritional value, has emerged as a prominent research focus in the field of food nutrition. Lactoferrin is widely distributed in raw milk and milk that has undergone low-temperature heat treatment during pasteurization, making its rapid and accurate detection crucial for ensuring the quality control of dairy products. An enzyme-linked immunosorbent assay-based analytical protocol has often been referred to for the detection of LF in real samples. Signal amplification was accomplished using the streptavidin-biotin system. Here, an automated magnetic beads-based sandwich chemiluminescence enzyme immunoassay (MBs-sCLEIA) system was developed for the quantification of lactoferrin in pasteurized milk. The MBs-sCLEIA system consists of an automated chemiluminescence-based analyzer and a lactoferrin MBs-sCLEIA assay kit. Notably, our proposed method eliminates the need for pretreatment procedures and enables the direct addition of milk samples, allowing for the automatic quantitative detection of lactoferrin within a rapid 17 min timeframe for up to eight samples simultaneously. The MBs-sCLEIA was linear over the range of 7.24-800 ng/mL and displayed a limit of detection (LOD) of 2.85 ng/mL. As its good recovery and CV values indicate, the method exhibited high precision and accuracy. Furthermore, it was verified that it was selective towards five additional common milk proteins. A good correlation was observed between the results from the MBs-sCLEIA and heparin affinity column-HPLC (r2 = 0.99042), which proves to be a useful and practicable way of conducting an accurate analysis of lactoferrin in dairy products.

"Milk on Ice": A detailed analysis of Ernest Shackleton's century-old whole milk powder in comparison with modern counterparts.

Whole milk powder (WMP) manufactured in New Zealand in 1907 was sent to the Antarctic continent with the Shackleton-led British Antarctic Expedition from 1907 to 1909. This powder was stored at ambient conditions at Shackleton's Hut at Cape Royds, Antarctica, for over 100 yr before a sample was collected on behalf of Fonterra by the Antarctic Heritage Trust. Having spent most of its existence both dried and in frozen storage, any deleterious reactions within the WMP would have been markedly retarded. The composition and some properties of the roller-dried Shackleton's WMP are reported along with those of 2 modern spray-dried New Zealand WMP. The Shackleton powder was less white and more yellow than the modern WMP and was composed of flakes rather than agglomerated particles, consistent with that expected of a roller-dried powder. Headspace analysis showed lipolytic and oxidative volatile compounds were present in the Shackleton WMP, indicting some deterioration of the milk either before powder manufacture or on storage of the finished product. On a moisture-free basis, the Shackleton WMP had higher protein, higher fat (with a markedly higher free fat level), higher ash, and a lower lactose level than the modern WMP. The lysine level was lower in the Shackleton WMP compared with the spray-dried powders, whereas the fatty acid composition was relatively similar. The sodium level was markedly higher in the Shackleton WMP compared with the spray-dried powder, which is probably due to the addition of an alkaline sodium salt to adjust the pH of the milk before roller drying. Lead, iron, and tin levels were markedly higher in the Shackleton WMP compared with the spray-dried powders, possibly due to the equipment used in powder manufacture and the tin-plated cases used for storage. The proteins in the Shackleton WMP were more lactosylated than in the spray-dried powders. The Shackleton WMP had a higher ratio of κ-casein A to B variants and a higher ratio of ß-lactoglobulin B to A variants than the spray-dried powders, whereas the αS1-casein, ß-casein, αS2-casein, and α-lactalbumin protein variants were similar in all powders. The total phospholipid content was markedly lower in the Shackleton WMP than the spray-dried powders, primarily due to a lower phosphatidylethanolamine concentration. The molecular species distributions within the phospholipid classes were generally similar in the 3 powders. Claims are sometimes encountered that the milk of today is different from that consumed by previous generations. However, this comparative study has shown that the Shackleton WMP was generally similar to modern WMP. Although differences in some components and properties were observed, these were attributable to the manufacturing equipment and processes used in the pioneering years of WMP manufacture.

Digestive properties and peptide profiles exhibited significant differences between skim camel milk and bovine milk powder after static in vitro simulated infant gastrointestinal digestion.

This study aims to analyze the differences in digestion properties and peptide profiles between the skim camel and bovine milk powder after static in vitro simulated infant gastrointestinal digestion. The hydrolysis degree of camel milk proteins exceeded by 13.18% that of bovine milk. The concentration and release rate of free amino groups in the camel milk digesta was higher than that of bovine milk powder, which was likely due to the higher ß-/αs-casein ratio and larger casein micelle size in camel milk. Camel milk powder presented higher ß-CN coverage and comparatively shorter bioactive peptides compared to bovine milk powder. The anti-inflammatory peptide KVLPVPQ displayed the highest abundance in camel milk powder. Outcomes of this study showed that camel milk proteins possessed superior digestibility and unique peptides, which outlined the potential nutritional implications of camel milk for infants.

Crystal size, a key character of lactose crystallization affecting microstructure, surface chemistry and reconstitution of milk powder.

Lactose crystallization during storage deteriorates reconstitution performance of milk powders, but the relationship between lactose crystallization and reconstitution is inexplicit. The objective of this study is to characterize crystalline lactose in the context of formulation and elucidate the complex relationship between lactose crystallization and powder functionality. Lactose in Skim Milk Powder (SMP), Whole Milk Powder (WMP) and Fat-Filled Milk Powder (FFMP) stored under 23 %, 53 % and 75 % Relative Humidity (RH) at 25  â„ƒ for four months was compared. Lactose, surface chemistry and microstructure of FFMP stored at 25 â„ƒ and 40 â„ƒ at 23 % to 75 % RH for four months were also analyzed and interpreted. At the same RH, FFMP crystallized in the same pattern as WMP. At 53 % RH, FFMP and WMP differentiated from SMP in terms of lactose morphology as well as the ratio between anhydrous α-lactose and anhydrous ß-lactose. Lactose remained amorphous at 23 % RH, crystallized predominantly to α/ß-lactose (1:4) at 40 to 58 % RH and to α-lactose monohydrate at 75 % RH. The crystallinity index was similar for all powders containing crystalline lactose. The estimated crystallite size increased from approx. 0.1 to 20 µm with increasing RH and temperature. When amorphous lactose crystallized into crystals below approx. 0.1 µm at 25 °C and 43 % RH, the microstructure and surface lipid were comparable to that of the reference powder. This powder reconstituted into a stable suspension system comparable to that of reference (well performing) powders. These results demonstrate that crystallite size is the key property linking lactose crystallization and reconstitution. Our finding thus indicates limiting crystallite size is important for maintaining desired product quality.

[Identifying animal-derived components in camel milk and its products by ultra-high performance liquid chromatography-tandem mass spectrometry].

A method for identifying specific peptide biomarkers of animal-milk-derived components in camel milk and its products was established using proteomics. Samples were prepared by defatting, protein extraction, and trypsin hydrolysis, and proteins and peptides were identified using ultra-high performance liquid chromatography-quadrupole/electrostatic orbitrap-high resolution mass spectrometry (UHPLC-Q/Exactive-HRMS) and Protein Pilot software. Twenty two peptide biomarkers from eight species (i.e., Camelus, Bos taurus, Bubalus bubalis, Bos grunniens/Bos mutus, Capra hircus, Ovis aries, Equus asinus, Equus caballus) were identified by comparing the basic local alignment search tool (BLAST) with the Uniprot database. Verification of these marker peptides were performed quantitatively using a UHPLC-triple-quadrupole mass-spectrometry (QqQ-MS) system by multiple reaction monitoring (MRM). The pretreatment method of casein in camel milk was optimized, such as defatting, protein precipitation, and re-dissolving buffer solution. The effects of various mass-spectrometry parameters, such as atomization gas, heating- and drying-gas flow rates, and desolvation-tube (DL) and ion-source-interface temperatures on ion-response intensity were optimized. Camel milk signature peptides were detected in a mixture of milk from other seven species to ensure specificity for the selected biomarker peptides. The signature peptides of seven other species were also detected in camel milk. No mutual interference between the selected biomarker peptides of the various species was observed. Adulterated camel milk and milk powder were also quantitatively studied by adding 0, 2.5%, 5%, 10%, 25%, 50%, 75%, and 100% bovine milk or goat milk to camel milk. Similarly, the same mass proportion of bovine milk powder or goat milk powder was added to camel milk powder. A quantitative standard curve for adulteration was constructed by plotting the peak areas of characteristic cow or goat peptide segments in each mixed sample against the mass percentage of the added adulterant. The adulteration standard curves exhibited good linearity, with correlation coefficients (r2) greater than 0.99. The limits of detection and quantification (LODs and LOQs, respectively) of the method were determined as three- and ten-times the signal-to-noise ratio (S/N). The minimum adulteration LODs of bovine milk and goat milk in camel milk were determined to be 0.35% and 0.49%, respectively, and the minimum LOQs were 1.20% and 1.69%, respectively. The minimum adulteration LODs of bovine milk powder and goat milk powder in camel milk powder were determined to be 0.68% and 0.73%, respectively, and the minimum LOQs were 1.65% and 2.45%, respectively. The accuracy of the adulteration quantification method was investigated by validating the quantitative detection results for 1∶1∶1 (mass ratio) mixtures of camel milk, bovine milk, and goat milk, as well as camel-milk powder, bovine milk powder, and goat-milk powder, which revealed that this method exhibits good linearity, strong anti-interference, high sensitivity, and good repeatability for adulterated liquid-milk/solid-milk-powder samples. The adulteration results for both liquid milk and milk powder are close to the theoretical values. Finally, 11 actual commercially available samples, including five camel-milk and six camel-milk-powder samples were analyzed, which revealed that only camel signature peptides were detected in 10 samples, while camel and bovine signature peptides were both detected in one camel-milk-powder sample. The ingredient list of the latter sample revealed that it contained whole milk powder from an unidentified source; therefore, we infer that the bovine signature peptides originate from the whole milk powder. These signature peptides also demonstrate the necessity and practical significance of establishing this identification method.

Rapid authentication of characteristic milk powders by recombinase polymerase amplification assays.

The authentication of dairy species has great significance for food safety. This study focused on a more rapid method for identifying major dairy species, and specific recombinase polymerase amplification (RPA)-based assays for cattle, goat, sheep, camel and donkey were developed. Through the developed RPA-based assays, goats and sheep could be simultaneously identified and bovine families could be differentiated. The performances of the RPA assays were validated using 37 milk powder samples, of which 16.2% (6/37) were suspected of being adulterated and 24.3% (9/37) were potentially at risk of being wrongly identified as adulteration. The effectiveness of the developed assays for crude DNA detection was also validated by a rapid nucleic acid extraction kit, and results showed that the presence of large amounts of protein and fat did not affect the qualitative results. Therefore, these assays could combine with the rapid nucleic acids extraction methods for being used in field detection.