Elucidating the Origin of Milk Products on the Chinese Market Using Hydrogen and Oxygen Stable Isotope Technique

Preference for foreign milk products is the cause of the economically motivated adulteration of milk products on the Chinese market. 42 milk samples from the United States of America, Canada, Southern China, Northern China, Australia and New Zealand were analyzed using δ2H and δ18O stable isotope technique to differentiate the origin of milk products. An isotope ratio mass spectrometer with a combination of a high-temperature conversion elemental analyzer, Thermo-Fisher was used. Statistical analysis was performed using one-way ANOVA. The study revealed δ2H and δ18O had a wide range of mean values: 13.86 to 22.25‰ and -82.86 to -28.5‰, respectively. There was a significant difference in the δ2H (n=7; F=20880, P=7.876E-43) and δ18O (n=7; F=1399.0; P=9.215E-29) composition of the milk samples from the different regions. It was observed that δ2H and δ18O composition is helpful in elucidating milk products from different regions on the Chinese market (P<0.05).


Introduction
Consumers in China are, for many reasons, asking for information about the food they are purchasing: the authenticity of the food and the geographical location of the food. Food adulteration is prominent in China and consumers are skeptical about the origin of food sold on the Chinese market [1]. Food adulteration affects the quality of food, which poses negative effects on consumers' nutrition and in some cases have led to adverse health effects including death [2]. According to Lu and Wu (2014), criminal cases relating to food safety that include food adulteration grew by 179.8% and 224.6% respectively; in 2011 and 2012 in China [3]. Thus, the need to study food fraud issues in China is necessary and timely. Milk is a major issue for the dairy industry in terms of adulteration. Significant profits on dairy items make them a prime target for adulteration [4]. Because Chinese consumers have a high preference for foreign milk products, they are the most vulnerable when it comes to milk adulteration and mislabeling. As it may not be obvious to detect the authenticity via physical observation, there is the urgent need for robust techniques that could provide unequivocal authentication outcome. Stable isotopes of hydrogen (H) and oxygen (O) in milk water are particularly essential for geographical origin assignment of food connected to regional climatic conditions because they are strongly latitude-dependent [5]. The δ18O content in milk water reflects the isotope composition of the ground water drunk by animals which in turn depends on the altitude, seasonality, total precipitation and distance from the sea [6]. This implies that it may be possible to discriminate among milk products that are obtained from different geographical origin using hydrogen and oxygen stable Isotopic signatures. The main aim of this study is to use the isotopic ratios of H and O within the milk, using mass spectroscopy, to reveal the true geographical origin of milk products on the Chinese market and tackle the mislabeling and deception done by local manufacturers.

Materials and Methods
Sample preparation and Statistical analysis: Forty-two milk samples were collected from different geographical origins, including the United States of America (USA), Canada (CA), Southern part of China (SC), Northern part of China (NC), Australia (AU), New Zealand (NZ). Each δ2H and δ18O composition of the samples (n=7 for each region) represent the mean of 3 replicates. Each liquid milk sample was obtained from genuine sources and was 100% authentic. The data obtained were analyzed using descriptive statistics and one-way analysis of variance (ANOVA) in the Excel Analysis ToolPak. A confidence level of 95% was accepted while an alpha value of 0.05 was used in the -one-way ANOVA.
Milk water: Moderate rennet (activity ≥105 U/g) obtained from the USA was added to pure milk and the samples were left overnight at room temperature. After, the milk water was collected by filtration and then frozen at -20 °C until analysis.
Standards: Stable isotope ratios are expressed in delta (δ) notation, was used to describe the isotopic difference between the sample and an international standard, which is defined by the equation.

Measurements:
The liquid milk samples were analyzed using an isotope ratio mass spectrometer (IRMS) operating in the continuous flow mode (Integra CN, Sercon, Cershire, UK) with a combination of a high temperature conversion elemental analyzer (TC/EA) attached Flash Element Analyzer 1112 HT-Delta V advantage, Thermo-Fisher for oxygen isotope analysis. For hydrogen isotopic analysis, the IRMS was equipped with an energy filter to suppress +4He ions on the attached Flash EA 1112 HT-Delta V advantage, Thermo-Fisher. The reactor consists of a glassy carbon tube with glassy carbon filling, ensuring that neither sample nor reaction gasses can get into contact with oxygen-containing surfaces. The reaction gasses are separated in an isothermal gas chromatograph, which is also part of the TC/EA. The gasses were admitted to the IRMS for isotope analysis. Measurements were made using calibration of the system with reference hydrogen and oxygen gas and reference standard material of VSMOW in regular repetitions.

Results
The mean δ2H and δ18O composition of milk samples are shown in Table 1 respectively. The range of the mean values for δ2H and δ18O are -82.86 to -28.50‰ and 13.86 to 22.25‰ respectively. The difference in the δ2H (F=20880; P= 7.876E-43; n=7) and δ18O (F=1399.0; P= 9.215E-29; n=7) composition of the samples were highly significant. Table 2 shows discriminating between the regions-of-origins of milk using oxygen and hydrogen stable isotope technique.

Discussion
Stable isotope analysis of is a powerful tool for provenance determination of food materials because isotopic compositions of the materials reflect many factors in the natural environment [7]. Renou et al. (2004) indicated stable isotope analysis can be a useful tool in showing a clear disparity between milk products from different regions [8]. Studies indicate that the H and O stable isotope values of animal tissue correlates with the isotopic composition of local precipitation [9]. The δ2H and δ18O composition in water consumed by animals shows a strong correlation with the δ2H and δ18O content present in animal products such as milk [10]. Particularly, Chesson et al. (2010) established that the δ2H and δ18O value of milk water show the isotopic composition of drinking water and water consumed from fresh forage, with minor deviations due to the contribution of food and atmospheric oxygen to the water body. According to Simpkins et al. (1999), the predominant influence on the fractionation of H and O isotopes is evaporation of water, which is reliant on climatic conditions such as humidity, temperature and rainfall. Moreover, the primary driver of the systematic geospatial patterns of H and O isotope ratios in precipitation is the preferential stripping of the heavy isotopes (i.e., 2H and 18O) from water vapor as ocean-saturated air masses move inland and across continents. Hence, at global to regional scales, the spatial variation in the isotopic composition of environmental water resources and human drinking water is consistent with the geographic patterns of precipitation isotopes. Studies indicate that as the δ2H and δ18O content of local water are related to the climatic and geographical features of an area, milk water with δ2H and δ18O can distinguish between milk produced in different areas [11][12][13].
From the present study, a very high significant difference in the δ2H and δ18O composition of milk water having a probability of 9.678E-6 and 9.215E-29 respectively (P< 0.05) were obtained from the regions under consideration ( Table 1).   [8]. Ritz et al. (2005) also explained that difference in the type of breed and difference in time elapsed since last pregnancy of the milk producing animal can also affect the δ18O composition [14]. Also, similar climatic conditions within the production regions may be the cause. This is because the content of δ18O depends on climatic conditions [5]. In a past study, stable isotope values were utilized to develop a new analytical approach enabling the identification of milk samples from different geographical origins [6]. The results they found were quite consistent with our results, which is that milk samples from the six different regions, the United States of America, Canada, Southern China, Northern China, Australia and New Zealand, could be easily discriminated and classified by δ2H and δ18O.
As a result of the extreme significant difference in the δ2H and δ18O composition of the milk samples obtained from the different geographical regions, fraudulent manufacturers cannot label milk products obtained from China as a milk obtained from a different geographical region. From our study, H and O stable isotope techniques may be useful in providing a unique discrimination in the origin of production of commercially distributed milk on the Chinese market. And thus could easily prevent the cheat of domestic pure milk to foreign pure milk or mislabeling pure milk.

Conclusions
China exports food to most parts of the world and it is not uncommon to find substandard products or mislabeled products in the Chinese market. Thus it is important that food exported to other countries from China are genuine and consumers are given what they have paid for. In this study, stable hydrogen and oxygen isotopic compositions (δ2H, δ18O) of 42 pure milk from various cultivated areas (the United States of America, Canada, Southern China, Northern China, Australia and New Zealand), were applied to discriminate the geographical origin of pure milk. It is clear from our discussions above that it is possible with varying degrees of certainty to determine the geographical origin of milk using δ2H, and δ18O.