Composition, physiochemical properties, nitrogen fraction distribution, and amino acid profile of donkey milk

This study investigated the changes in chemical composition, nitrogen fraction distribution, and AA profile of milk samples obtained during lactation from the Jiangyue breed of donkey in Northwest China. Results showed that donkey milk contained 9.53% total solids, 1.57% protein, 1.16% fat, 6.33% lactose, and 0.4% ash on average, which is more similar to mare and human milk than to the milk of other mammals. Throughout the lactation investigated, pH and density were constant, protein and ash content showed an apparent negative trend (an increase in lactose content during 120 d postpartum, followed by a decrease), fat content exhibited wide variability, and variations in the content and percentage of whey protein, casein, and AA were small. The casein to whey protein ratio of 52:37 was between the lower value of human milk and the higher value of cow milk. Sodium dodecyl sulfate-PAGE results demonstrated that donkey milk is rich in β-lactoglobulin and lysozyme. The percentages of 8 essential AA in protein of donkey milk were 38.2%, higher than those of mare and cow milk; donkey milk also had higher levels of serine (6.2%), glutamic acid (22.8%), arginine (4.6%), and valine (6.5%) and a lower level of cystine (0.4%).     

Fermented beverage emulsion based on donkey milk with sunflower oil

Sunflower oil was added to donkey milk to increase the low energetic intake and improve both texture and healthy characteristics. This milk‐based emulsion was fermented using a binary association of two strains of Streptococcus thermophilus showing distinctive abilities. One strain (St 907) produced ropy exopolysaccharide (EPS), whereas the other strain (St 563) produced folic acid. Fermentation was stopped at pH 5.0 to prevent whey separation. The refrigerated product was stable at least for 20 days and showed improved viscoelastic properties due to the EPS combined with the oil droplets. The volatile profile was constituted of ketones, aldehydes, alcohols and fatty acids, mainly originated from microbial fermentation, whereas few other compounds suggested the presence of oxidative processes. The folic acid content in the fermented beverage was increased by more than 10 times (2.03 ± 0.17 μg/100 mL) with respect to that found in donkey milk (0.16 ± 0.03 μg/100 mL).     

响应面法优化风味驴乳酸奶发酵工艺的研究

在驴乳中添加牛乳粉制作酸奶,通过单因素试验和响应面优化法研究了风味驴乳酸奶最佳发酵工艺。结果表明,以牛乳粉添加量、发酵温度和发酵时间为自变量,以感官评价为因变量,响应面法优化出风味驴乳酸奶的最佳发酵工艺为：牛乳粉添加量10%、发酵温度42 ℃、发酵时间5.5 h。在此优化条件下,风味驴乳酸奶口感细腻爽滑,酸甜适口,有浓郁的驴乳香味与滋味,感官评分达到92分,与预测值（90.85分）相差较小。产品的理化及微生物指标均符合相关国标要求。     

A preliminary investigation into the unsaponifiable fraction of donkey milk: Sterols of animal origin,phytosterols, and tocopherols

We investigated the main sterols, phytosterols, and the α- and γ-tocopherol content in donkey milk during the first 2 mo of lactation. Cholesterol was the main sterol in milk (mean ± standard deviation = 0.97 ± 0.443 g/100 g of fat). Lanosterol was the main minor sterol of animal origin, followed by desmosterol (0.003 ± 0.001 and 0.001 ± 0.001 g/100 g of fat, respectively). Of the phytosterols, β-sitosterol was the main sterol of vegetal origin in donkey milk (0.005 ± 0.002 g/100 g of fat), but lower levels of campesterol, brassicasterol, and stigmasterol were also recorded. Mean levels of α- and γ-tocopherol were 0.01 ± 0.007 and 0.003 ± 0.001 g/100 g of fat, respectively. We observed no significant changes in sterol or tocopherol content during the first 2 mo of lactation. The presence of lanosterol in donkey milk is of particular interest, because lanosterol is a potential drug and has important physiological effects. The presence of phytosterols, which are considered nutraceutical molecules, enhances the nutritional quality of donkey milk fat for consumers.     

Short communication: Technological and seasonal variations of vitamin D and other nutritional components in donkey milk

Vitamin D is an essential nutrient that plays a crucial role in calcium homeostasis and bone metabolism and also acts as a hormone. Although several studies on the content of vitamin D in bovine milk have been conducted, little information is available regarding donkey milk. In the context of the nutritional assessment of donkey milk, the aim of this study was to assess the vitamin D content in donkey milk and its chemical profile, with particular reference to seasonal and technological modifications after pasteurization. The study was conducted on a dairy farm that produces donkey milk for human consumption located in central Italy. At sampling time, an aliquot of total bulk milk production was sampled before and after pasteurization (63°C for 30 min without homogenization) with a total of 20 raw and 20 pasteurized milk samples. The samples were collected for 10 mo, every 15 d, from May to February 2017. All the samples were analyzed for the chemical composition and vitamin D₂ and D₃ content by HPLC after saponification. The donkey milk analyzed showed a higher average vitamin D content (raw milk: vitamin D₂ = 1.68, vitamin D₃ = 0.60 μg/100 mL; pasteurized milk: vitamin D₂ = 1.38, vitamin D₃ = 0.30 μg/100 mL) than reported for bovine and human milk. The results of the effect of pasteurization on milk did not highlight significant differences in the total content of vitamin D. However, vitamin D₃ has a poor thermal stability, which led to a significant reduction in content in pasteurized milk compared with raw milk. The total vitamin D content of donkey milk did not show significant variations between seasons; however, a higher concentration of vitamin D₃ was found in spring and summer. In conclusion, raw and pasteurized donkey milk showed a high content of vitamin D, which could be useful in meeting the deficiencies of this vitamin in humans. Further investigations are needed to improve the vitamin D content in donkey milk by increasing its endogenous synthesis or its transfer in milk and to clarify other variability factors.     

模糊数学评价法优化驴乳奶啤稳定性的预处理参数及香气成分分析

基于模糊数学评价法对驴乳奶啤稳定性预处理参数初步优化,结合气相色谱-质谱联用（GC-MS）技术确定驴乳奶啤的最佳预处理参数,通过与牛乳奶啤香气成分差异化对比,探究驴乳奶啤的香气组成。结果表明,最佳预处理参数为单甘脂添加量0.10%、海藻酸丙二醇酯添加量0.15%、果胶添加量0.20%。在此最佳条件下,5号驴乳奶啤综合评分最高（84.20分）,稳定性系数、离心沉淀率、黏度分别为（98.23±0.17）%、（5.21±0.14）%、（37.13±0.08） mPa·s。从牛乳奶啤及5号、9号驴乳奶啤中共检测出79种香气化合物,包括醇类11种、酯类25种、醛类8种、酸类12种、酮类6种、苯类2种、烷烃类12种、萜烯类2种、其他类1种,其中醇类、酯类和酸类挥发性化合物相对含量较高,对牛乳奶啤及5号、9号驴乳奶啤共有的18种香气成分进行主成分分析,经综合评价得出5号驴乳奶啤香气品质最佳,GC-MS分析结果与综合模糊评判结果一致。     

驴乳中蛋白质组分的分离纯化与鉴定

目的：分离纯化并鉴定驴乳乳清中蛋白质组分,为进一步研究驴乳用于辅助治疗疾病,以及为作为人乳替代品提供参考。方法：采用DEAE-52 离子交换层析和Sephadex G-100 凝胶层析分离纯化驴乳乳清中蛋白质组分,并通过十二烷基硫酸钠--聚丙烯酰胺凝胶电泳法和高效凝胶渗透色谱法对所纯化蛋白质进行鉴定。结果：与牛乳乳清中蛋白组分相对比,发现驴乳乳清中存在3 种未知蛋白质,分子质量分别为32、70.1、72.2kD。结论：驴乳中除了含有与牛乳相似的营养成分外,还具有其他生物活性成分,它们可能是一些保护性蛋白,在机体的抗病机制方面起着重要作用。     

Chemical composition,protein fraction and fatty acid profile of donkey milk during lactation

The chemical composition, fatty acid and protein fraction of donkey milk samples from domestic (Arcadian) breed were assessed from the 30th day to the 210th day of lactation. Total fat, proteins and minerals (Ca, Mg, K and P) were affected by the stage of lactation. Fatty acids were significantly influenced, with a decrease of 31.4% in saturated fatty acids, an increase of 53.8% in unsaturated fatty acids, a concomitant elevation of polyunsaturated fatty acids and a low n-6/n-3 ratio (2:1). Analysis by sodium dodecylsulphate-polyacrylamide gel electrophoresis found, on average, 42.8% casein and 57.2% whey protein. The concentration of lysozyme was between 1.20 and 2.54 mg mL⁻¹, while the highest values were detected by the lysoplate method. Finally, low microbial content and somatic cells were also found in donkey milk with overall average log 4.4 cfu mL⁻¹ and log 4.8 cells mL⁻¹, respectively.     

Antiviral activity of donkey milk protein fractions on echovirus type 5

Antiviral activity of Ragusano donkeys' milk proteins was investigated for the effect on echovirus type 5, known to infect the gastrointestinal tract of humans. Three protein fractions were tested; casein (CN), whey protein (WP) and a low molecular whey protein fraction (LWP; <30,000 Da). The antiviral activity of WP and LWP was tested on echovirus type 5 at three concentrations (1, 5 and 10 mg mL^?1); CN was assessed only at the lower concentration. All donkey milk protein fractions showed significant inhibition on virus replication at the concentration of 1 mg mL^?1, and both WP and LWP fractions showed significant inhibition on the virus replication at all concentrations tested. The strongest antiviral effect was observed for the WP fraction. These findings show that the different whey proteins in donkey milk, probably acting in synergy, exert antiviral activity on echovirus 5 and might contribute to prevent gastrointestinal virus infections in humans.     

Validation of a gold standard method for iodine quantification in raw and processed milk,and its variation in different dairy species

Adequate milk consumption significantly contributes to meeting the human iodine recommended daily intake, which ranges from 70 µg/d for infants to 200 µg/d for lactating women. The fulfilment of iodine recommended daily intake is fundamental to prevent serious clinical diseases such as cretinism in infants and goiter in adults. In the present study iodine content was measured in raw and processed commercial cow milk, as well as in raw buffalo, goat, sheep, and donkey milk. Iodine extraction was based on 0.6% (vol/vol) ammonia, whereas iodine detection and quantification were carried out through an inductively coupled plasma mass spectrometer analyzer. Among processed commercial cow milk, partially skimmed pasteurized milk had the greatest iodine content (359.42 µg/kg) and raw milk the lowest (166.92 µg/kg). With regard to the other dairy species, the greatest iodine content was found in raw goat milk (575.42 µg/kg), followed by raw buffalo (229.82 µg/kg), sheep (192.64 µg/kg), and donkey milk (7.06 µg/kg). Repeatability of milk iodine content, calculated as relative standard deviation of 5 measurements within a day or operator, ranged from 0.96 to 1.84% and 0.72 to 1.16%, respectively. The overall reproducibility of milk iodine content, calculated as relative standard deviation of 45 measurements across 3 d of analyses and 3 operators, was 4.01%. These results underline the precision of the proposed analytical method for the determination of iodine content in milk.     

Development of a real-time PCR assay for the detection of cow and donkey milk

A real-time PCR allelic discrimination TaqMan assay based on the analysis of a single nucleotide polymorphism enabling the differentiation of cow (Bos taurus) and donkey (Equus asinus) milk was developed. Specific primers and probes were designed on the mitochondrial cytochrome c oxidase subunit I gene. The primers were designed upstream and downstream the chosen diagnosis site in a conserved region. Two probes were designed to specifically hybridise to B. taurus and E. asinus sequences. The test allowed the discrimination of bovine and donkey DNA in all blood and pure milk samples giving an unambiguous result plot of rapid and easy interpretation. The detection threshold was 2?% of cow milk in donkey milk. The applicability of the method to matrices containing degraded DNA was demonstrated by analysing samples of raw donkey and cow milk autoclave-treated (121?°C for 15?min). Finally, the assay when applied to milk samples collected from the retail trade has confirmed the species indicated in the label. Furthermore, the assay represents a potentially valuable diagnostic tool for species identification in dairy products for allergic people.     

Lysozyme activity in donkey milk

Lysozyme activity of donkey milk in relation to the lactation stage, age of the animals and pasteurisation treatment was studied. Raw and pasteurised bulk milk samples were collected monthly for one year and were analysed in relation to lysozyme activity. Individual raw milk samples were collected from 12 jennies, at three lactation stages: three, six and nine months. The results showed that the lactation stage and the age of the animals influenced the lysozyme activity of donkey milk. The highest lysozyme activity values were found at early lactation and in animals older than 15 years. We confirm that low-temperature long-time pasteurisation of donkey milk does not reduce the activity of this enzyme. In addition, calcium, ash and some fatty acids, and, in particular, the saturated fatty acids, are linked to a higher lysozyme activity of donkey milk at early lactation.     

Production of cheese from donkey milk as influenced by addition of transglutaminase

Donkey milk is characterized by low contents of total solids, fat, and caseins, especially κ-casein, which results in formation of a very weak gel upon renneting. The objective of this study was to evaluate the effect of fortification of donkey milk with microbial transglutaminase (MTGase) for cheesemaking in relation to different enzyme addition protocols (patterns, PAT). Four independent trials were performed using MTGase (5.0 U/g of milk protein) according to the following experimental patterns: control (no MTGase addition); MTGase addition (40°C) 15 min before starter inoculation (PAT1); addition of MTGase to milk simultaneously with starter culture (40°C) (PAT2); and MTGase addition simultaneously with rennet (42°C) in acidified milk (pH 6.3) (PAT3). Evolution of pH during acidification, cheesemaking parameters, and proximal composition and color of cheese at 24 h were recorded. The protein fractions of cheese and whey were investigated by urea-PAGE and sodium dodecyl sulfate-PAGE. Addition of MTGase had no significant effect on moisture, protein, fat, or cheese yield. The addition of MTGase with rennet (PAT3) improved curd firmness compared with the control. Among the different patterns of MTGase addition, PAT3 reduced gel formation time, time between rennet addition and cheese molding, and weight loss of cheese at 24 h. The PAT3 treatment also resulted in the lowest lightness and highest yellowness color values of the cheese. Sodium dodecyl sulfate-PAGE of cheeses revealed that MTGase modified the protein pattern in the high-molecular-weight zone (range 37–75 kDa) compared with the control. Of the MTGase protocols, PAT3 showed better casein retention in cheese, as confirmed by the lanes of α- and β-caseins in the electropherogram of the whey, which was subtler for this protocol. In conclusion, MTGase may be used in cheese production from donkey milk to improve curd firmness; MTGase should be added simultaneously with the rennet.     

Size exclusion and reversed-phase high-performance liquid chromatography/UV for routine control of thermal processing of cows' and donkey milk major proteins

Cows' and donkey milks (raw and thermally processed) and respective whey were analysed for quantification of major proteins. Two different chromatographic approaches, size exclusion (SE-HPLC) and reversed-phase high performance liquid chromatography (RP-HPLC) both coupled to UV detection were used. Usefulness of these methods for routine control of the effect of thermal processing was evaluated. The external standard method was used to calibrate the SE-HPLC and RP-HPLC systems. Concerning quantification of β-lactoglobulin (β-lg), α-lactalbumin (α-la), lysozyme (lys), and total casein (cn), no significant differences between results obtained by SE-HPLC and by RP-HPLC (t-test, P>0·05) were observed for raw milks and whey. Heating of cows' milk promoted aggregation of denatured proteins as observed by SE-HPLC, whereas α-la and β-lg from donkey milk were stable to thermal processing at 100 °C (5 min). Lys was quantified in donkey raw milk and whey however, in thermally processed donkey milk lys was denatured and could not be quantified by HPLC.     

Invited review: Human,cow, and donkey milk comparison: Focus on metabolic effects

Milk is an important food of the daily diet. Many countries include it in their dietary recommendations due to its content in several important nutrients that exert beneficial effects on human health. Human milk is a newborn's first food and plays an important role in the growth, development, and future health of every individual. Cow milk is the type of milk most consumed in the world. However, its relatively high content of saturated fats raises concerns about potential adverse effects on human health, although epidemiological studies have disproved this association. Indeed, dairy consumption appear to be linked to a lower risk of mortality and major cardiovascular disease events. In the last few years many researchers have begun to focus their attention on both the production and quality of cow milk as well as the analysis of milk from other animal species to evaluate their effect on human health. The need to investigate the composition and metabolic effects of milk from other animal species arises from the adverse reactions of individuals in several groups to certain components of cow milk. It has emerged that donkey milk compared with that of other animal species, is the nearest to human milk and an excellent substitute for it. Milk from various animal species shows substantial differences in nutritional composition and distinct metabolic effects. In this review, we discussed the main compositional features and metabolic effects of 3 types of milk: human, cow, and donkey milk.     

Investigation of donkey milk bacterial diversity by 16S rDNA high-throughput sequencing on a Cyprus donkey farm

The interest in milk originating from donkeys is growing worldwide due to its claimed functional and nutritional properties, especially for sensitive population groups, such as infants with cow milk protein allergy. The current study aimed to assess the microbiological quality of donkey milk produced in a donkey farm in Cyprus using culture-based and high-throughput sequencing techniques. The culture-based microbiological analysis showed very low microbial counts, whereas important food-borne pathogens were not detected in any sample. In addition, high-throughput sequencing was applied to characterize the bacterial communities of donkey milk samples. Donkey milk mostly composed of gram-negative Proteobacteria, including Sphingomonas, Pseudomonas, Mesorhizobium, and Acinetobacter; lactic acid bacteria, including Lactobacillus and Streptococcus; the endospores forming Clostridium; and the environmental genera Flavobacterium and Ralstonia, detected in lower relative abundances. The results of the study support existing findings that donkey milk contains mostly gram-negative bacteria. Moreover, it raises questions regarding the contribution of (1) antimicrobial agents (i.e., lysozyme, peptides) in shaping the microbial communities and (2) bacterial microbiota to the functional value of donkey milk.     

Distribution of selected trace elements in the major fractions of donkey milk

The aim of this study was to evaluate the concentrations of Zn, Cu, Mn, Se, Mo, Co, Li, B, Ti, Cr, Rb, Sr, Cd, and Pb in donkey milk and their distribution in major milk fractions (i.e., fat, casein, whey proteins, and aqueous phase). Individual milk samples were provided by 16 clinically healthy lactating donkeys. Subsequent centrifugation, ultracentrifugation, and ultrafiltration were carried out to remove fat, casein, and whey proteins to obtain skim milk, a supernatant whey fraction, and the aqueous phase of milk, respectively. Concentrations of the elements were measured in whole milk and fractions by inductively coupled plasma-mass spectrometry, and the concentrations associated with fat, casein, and whey proteins were then calculated. The effect of removal of fat, casein, and whey proteins was determined by repeated-measures ANOVA. The fat fraction of donkey milk carried a small (～4.5% to 13.5%) but significant proportion of Mo, Co, Ti, Cr, and Sr. The casein fraction in donkey milk carried almost all milk Zn, a majority of Cu and Mn, and most of Mo, Ti, and Sr. Relevant proportions, between 20% and 36%, of Se, Co, and Cr were also associated with caseins. The majority of Se, Co, Li, B, Cr, and Rb, and relevant proportions of Mn, Mo, Ti, and Sr were found in soluble form (ultracentrifuged samples) and distributed between whey proteins and the aqueous phase of milk (ultrafiltered samples). Whey proteins in donkey milk carried the majority of milk Se and Co. All Li and B was present in the aqueous phase of milk, which also contained most Rb and Cr, and 17% to 42% of Mn, Se, Mo, Co, Ti, and Sr.     

Production and evaluation of some physicochemical parameters of peanut milk yoghurt

The peanut milk for yoghurt production was prepared by fortifying peanut milk (∼12 g/100 g total solids) with 4 g/100 g skimmed milk powder. The final product was subjected to physicochemical analysis using cow milk yoghurt (CMY) as a control throughout the study. Peanut milk yoghurt (PMY) had higher protein content, fat, water holding capacity and lower susceptibility to Syneresis than CMY. PMY had lower lactose level (1.73 g/100 ml) compared to CMY (4.93 g/100 ml). Generally both PMY and CMY had high mineral composition and contained high amounts of essential amino acids. PMY also contained a higher proportion of unsaturated fatty acids than saturated fatty acids as compared to CMY. Therefore, in terms of fatty acid composition, PMY could be considered to be more health promoting than CMY. Sensory evaluation revealed that though PMY had better sensory texture scores than CMY, its sensory appearance, flavor and overall acceptability scores were lower than those of CMY.     

不同来源乳中α-乳白蛋白、β-乳球蛋白和乳铁蛋白含量分析

本研究通过高效液相色谱-质谱联用法(HPLC-MS)对不同种乳中α-乳白蛋白、β-乳球蛋白和乳铁蛋白的含量进行测定和比较。结果显示,不同来源乳中α-乳白蛋白、β-乳球蛋白、乳铁蛋白和总蛋白存在一定差异。羊乳中乳清蛋白平均值为0.385 mg/100 g,低于牛乳和牦牛乳;羊乳中乳铁蛋白含量最高,均值为4.43 mg/100 g,最大值9.90 mg/100 g,是优质的乳铁蛋白来源;牦牛乳总蛋白含量(均值3.61%),明显高于牛乳(均值3.22%)和羊乳(均值2.89%);牛奶中乳清蛋白量在三类乳中最高。     

驴脂组成成分及其油脂的品质指标分析

研究了驴脂的元素谱库、营养成分组成,测定了驴脂提取分离产物驴油的品质指标。以电感耦合等离子体质谱技术(ICP-MS)对驴脂中37种金属元素进行扫描分析,形成样本的元素谱库,结果表明驴脂中各金属元素均未达到检出限。以多质量分析器组合式质谱仪对驴脂进行MSn级扫描,最终获得驴脂中主要成分信息,发现驴脂主要成分中包含16种氨基酸及其衍生物类、11种有机酸、46种脂质以及糖类、维生素类等多种成分。采用超声辅助溶剂提取法得到的驴油相对密度为0. 908,水分含量为0. 68%,酸价(KOH)为0. 58 mg/g,过氧化值为0. 018 g/100 g,碘值(I)为85g/100 g。研究储存温度对驴油过氧化值的影响,结果表明驴油常温下即可储存。驴脂可作为食品、化妆品的研发原料,具有广阔的应用前景。