A characteristic fatty acid fingerprint of human milk and its application in authenticity identification

Abstract

This invention discloses a characteristic fatty acid fingerprint spectrum of human milk and its application in authenticity identification, belonging to the field of food analysis technology. The milk characteristic fatty acid fingerprint spectrum of this invention uses C8:0 and C14:1 in human milk as the basis for identification. cis-9 C15:0, C15:0 before , C18:2 c9t11(CLA n7), C18:2 c9c12(LA), C20:1 cis-11 Based on eight characteristic fatty acids (C22:6, C4, C7, C10, C13, C16, C19, and DHA), a standard human milk fingerprint spectrum is constructed based on the ratios of C8:0 / LA, C8:0 / DHA, C14:1 cis-9 / C20:1 cis-11, C15:0 / DHA, C15:0 anteiso / C20:1 cis-11, and CLA / LA. The content of characteristic fatty acids in the test sample is measured, and the corresponding ratios are calculated and compared with the standard human milk fingerprint spectrum. If any ratio exceeds the characteristic range, the sample is determined to be not entirely human milk. This invention's human milk characteristic fatty acid fingerprint spectrum utilizes six standard indicators for multi-dimensional judgment, enabling rapid and accurate identification of human milk authenticity. It only requires measuring fatty acid content, making it simple and quick. The fingerprint spectrum has strong applicability for authenticity identification, allowing for sample-free determination using historical data and literature data, providing strong traceability.

Description

Technical Field

[0001] This invention belongs to the field of food analysis technology, specifically relating to a characteristic fatty acid fingerprint spectrum of human milk and its application in authenticity identification. In the application of human milk authenticity identification, the sample to be tested is compared with the standard human milk fingerprint spectrum, which can effectively identify the counterfeit or adulterated human milk sample. Background Technology

[0002] Milk is the primary dietary source for mammalian offspring, providing a range of nutrients beneficial to human health, including lipids, proteins, bioactive peptides, minerals, and vitamins (Liu et al., 2018). my country boasts a rich variety of dairy products, including common cow's milk (Holstein and Jersey milk), as well as specialty milks such as goat's milk, buffalo milk, and yak milk. Compared to animal milk, human milk has always been considered the most beneficial food for the growth and development of human infants. Therefore, research on the nutritional value of human milk has become an important benchmark for evaluating the nutritional quality of dairy products and a reference for infant formula formulation. However, due to current ethical concerns, obtaining human milk samples is extremely difficult, which to some extent restricts research related to human milk, such as in medicine, physiology, food science, and forensic medicine. In this context, ensuring the authenticity of human milk samples is particularly important. The substitution of animal milk for human milk will lead to deviations in the direction of relevant scientific research and investigation.

[0003] Currently, milk authenticity can be determined by detecting proteins or genes in milk. Sharma et al. (2021) used non-immunoglobulin antigens in milk as indicators to identify milk adulteration as low as 5% in buffalo milk using an immunoassay method. Malo et al. (2018) used immunoglobulins in milk as indicators to identify milk adulteration as low as 1% in goat milk, sheep milk, and buffalo milk using an immunoassay method. Liu et al. (2019) used casein in milk as an indicator to identify milk adulteration as low as 0.07% in goat milk using an immunoassay method, while Ren et al. (2014) also used casein in milk to identify milk adulteration as low as 1% in yak milk. Trimboli et al. (2019) used capillary electrophoresis to detect α-lactalbumin content in milk, and using α-lactalbumin as an indicator, they were able to identify milk adulteration as low as 1% in buffalo milk and quantify adulteration levels above 3.1% in buffalo milk. Chen et al. (2016) used proteomics to qualitatively and quantitatively analyze cow's milk adulteration in goat's or sheep's milk using β-lactoglobulin as an indicator. De et al. (2011) used mitochondrial DNA circular fragments as target sequences and amplified 126bp and 226bp specific PCR products of cow's milk and buffalo milk, respectively, using species-specific DNA sequences to identify cow's milk adulteration down to 0.01%. However, processing methods such as homogenization and heat treatment of dairy products are highly destructive to proteins and genes, resulting in a wide range of variations in protein content and numerous interfering factors. Therefore, identification methods based on protein characteristics for species differences have low sensitivity and are prone to false positives, while identification methods based on species-specific gene differences, although highly sensitive, are time-consuming, expensive, and have a high application threshold, making them difficult to popularize.

[0004] In addition, the authenticity of dairy products can also be determined through cluster analysis of specific nutrients in milk. Pereira et al. (2020) determined the total fat and protein content in cow's milk and goat's milk using near-infrared spectroscopy and established a PLS-DA model that can identify cow's milk adulterated with a content as low as 1.0154 g / 100 g in goat's milk. Sen et al. (2021) determined the total fat, protein, lactose, and non-fat solids content in cow's milk, buffalo milk, and goat's milk using Fourier transform infrared spectroscopy and established an OPLS-DA model that can identify two types of milk mixtures with a mixing level higher than 5%: cow's milk-goat's milk and cow's milk-buffalo milk, with discriminant analysis accuracy rates of 93% and 91%, respectively. Zhang et al. (2018) determined the fatty acid content in cow's milk and mare's milk using gas chromatography and used PCA, SIMCA, and PLS models to analyze mixed samples of cow's milk and mare's milk, demonstrating the feasibility of using fatty acid fingerprinting for the authenticity of specialty milk products and the ability to identify cow's milk adulteration with a content as low as 10% in mare's milk.

[0005] Raw milk contains a rich variety of fatty acids, with significant differences in their composition and content among different species. Fatty acid fingerprinting, which summarizes the composition and content characteristics of all fatty acids in milk, is a species-specific identification technique that is more accurate and reliable for authenticating dairy products. However, current fatty acid determination results are mostly presented as relative contents, requiring area normalization quantification of all fatty acids in milk (Liu et al., 2018), which significantly increases analysis time. Selecting characteristic fatty acid ratios not only simplifies the analytical process and improves the efficiency of authenticity identification but also maximizes the differences, thereby improving the accuracy of authenticity identification. Studies have found that the fatty acid ratios C10:0 / C8:0, C12:0 / C10:0, C14:0 / C12:0, and C14:0 / C18:1 can distinguish between the adulteration of butter and lard in milk fat (Anmat, 2011). However, the method proposed by Rebechi et al. (2016) can only identify the adulteration of butter or lard in milk fat with a content of more than 15%, indicating insufficient detection sensitivity.

[0006] While the aforementioned methods can identify adulterated dairy products to some extent, their application to the authenticity of human milk has limitations. For example, pretreatment processes such as separation and extraction are required before testing, leading to cumbersome operation, long processing time, high analytical costs, low sensitivity, and poor applicability. Existing methods for authenticating human milk are poorly researched, and there is no method specifically designed for human milk authenticity with strong applicability and traceability. Therefore, a method for authenticating human milk and its products that is accurate, easy to operate, applicable, and traceable is urgently needed. Summary of the Invention

[0007] To overcome the shortcomings of existing methods for authenticating dairy products, particularly the lack of a suitable and traceable method for authenticating human milk, this invention aims to provide a set of characteristic ranges for the ratios of characteristic fatty acids in human milk. Based on these ranges, a fingerprint of human milk is constructed and applied to the authenticity identification of human milk and related products.

[0008] The inventive concept of this invention is as follows: Compared with animal milk such as cow's milk, buffalo milk, yak milk, goat's milk, donkey milk, and camel milk, human milk has a higher content of fatty acids such as LA, DHA, and C20:1c11, while the content of fatty acids such as C8:0, C15:0, C15:0 anteiso, C14:1c9, and CLA is lower. This invention utilizes extensive experimental data and background investigation data from previous studies to statistically analyze and screen various fatty acid ratios, identifying several ratios with extremely low values ​​in human milk and significant differences from those in other animal milks as characteristic fatty acid ratios. For each characteristic fatty acid ratio, the characteristic range in human milk is calculated using AVG (mean) ± 3 × SD (standard deviation), with a confidence level of 99%, and a radar chart is created to form a fingerprint spectrum. The characteristic fatty acid ratios of the milk sample to be tested are measured and calculated, and then compared with the aforementioned fingerprint spectrum for authenticity verification.

[0009] The specific technical solution of this invention is as follows:

[0010] A fingerprint of characteristic fatty acids in human milk, wherein the characteristic fatty acids in human milk are: C8:0, C14:1cis-9, C15:0, C15:0anteiso, C18:2c9t11 (CLA), C18:2c9c12 (LA), C20:1cis-11, and C22:6c4c7c10c13c16c19 (DHA).

[0011] A characteristic fatty acid fingerprint of human milk is constructed based on the ratios of six fatty acids in human milk: C8:0 / LA, C8:0 / DHA, C14:1cis-9 / C20:1cis-11, C15:0 / DHA, C15:0anteiso / C20:1cis-11, and CLA / LA. The ratio range of the characteristic fatty acids is as follows:

[0012]

[0013]

[0014] A method for identifying the authenticity of human milk using the aforementioned characteristic fatty acid fingerprint spectrum includes the following steps:

[0015] S1: Determine the content of characteristic fatty acids in the test sample and calculate the corresponding ratio of characteristic fatty acids in human milk;

[0016] S2: Compare the corresponding fatty acid ratios calculated in step S1 with the characteristic fatty acid fingerprint of human milk. If any characteristic fatty acid ratio exceeds the characteristic range, i.e. outside the confidence range of the radar chart, the sample is determined to be not completely human milk and is counterfeit or adulterated.

[0017] Preferably, the determination of the characteristic fatty acid content in the human milk sample to be tested in step S1 is performed by gas chromatography or gas chromatography-mass spectrometry.

[0018] Preferably, the results of calculating the corresponding fatty acid ratios in step S1 are expressed as relative values ​​(% of total fatty acids).

[0019] Preferably, the result of calculating the corresponding fatty acid ratio in step S1 is expressed as an absolute value (mg / L milk).

[0020] Compared with the prior art, the beneficial effects of this invention are as follows:

[0021] (1) Fingerprint characteristics (exclusivity): The characteristic fatty acid ratios selected in this invention exhibit obvious fingerprint characteristics. Compared to other animal milks, the above ratios are all extremely small. The six indicators are judged from multiple dimensions, enabling rapid and accurate differentiation from other animal milks.

[0022] (2) Simple and quick: Compared with immunological methods for determining proteins or nucleic acids, the determination of fatty acid content is simpler and can be completed in 4 to 6 hours. More importantly, fatty acids are a basic nutritional indicator and a commonly tested indicator for milk samples. Therefore, the conclusion of the authenticity of human milk can be obtained simply by analyzing the existing test results using this method.

[0023] (3) High applicability: Compared with immunoassay methods for determining proteins or nucleic acids, the determination of fatty acid content has a low technical threshold and low cost. The results can be determined by gas chromatography or gas chromatography-mass spectrometry, or by a rapid infrared determination device.

[0024] (4) Strong traceability: The method for identifying the authenticity of human milk based on characteristic fatty acid fingerprinting of the present invention can be determined by data, and can be used to make determinations without samples for historical data, literature data, etc. Attached Figure Description

[0025] Figure 1 This is a radar chart showing the characteristic fatty acid ratio range of the human milk characteristic fatty acid fingerprint spectrum of the present invention. Detailed Implementation

[0026] To enable those skilled in the art to better understand the present invention, it will now be further described in conjunction with specific embodiments. The following examples are for illustrative purposes only and are not intended to limit the scope of the invention.

[0027] Example 1

[0028] Based on eight characteristic fatty acids in human milk (C8:0, C14:1cis-9, C15:0, C15:0anteiso, C18:2c9t11 (CLA), C18:2c9c12 (LA), C20:1cis-11, and C22:6c4c7c10c13c16c19 (DHA), a standard human milk characteristic fatty acid fingerprint spectrum was constructed based on six ratios: C8:0 / LA, C8:0 / DHA, C14:1cis-9 / C20:1cis-11, C15:0 / DHA, C15:0anteiso / C20:1cis-11, and CLA / LA. The range of these ratios is shown in Table 1.

[0029] Table 1: Characteristic range of fatty acid ratios in human milk

[0030]

[0031]

[0032] A radar chart of the characteristic fatty acid ratio ranges of human milk was plotted based on the aforementioned characteristic fatty acid ratio ranges; see appendix. Figure 1 .

[0033] Example 2

[0034] Fifty-nine samples of Holstein cow milk were taken for verification analysis. The content of characteristic fatty acids in the samples was determined, the corresponding fatty acid ratios were calculated, and compared with the standard human milk fingerprint spectrum constructed in Example 1. If any characteristic fatty acid ratio exceeded the characteristic range, that is, outside the confidence range of the radar chart, the sample was determined to be not completely human milk and to be counterfeit or adulterated. The results are shown in Tables 2-1 and 2-2.

[0035] Table 2-1 Characteristic fatty acid content (% total fatty acids, n=59) of Holstein cow milk adulterated with human milk

[0036]

[0037]

[0038]

[0039] Note: The DHA content in animal milk is extremely low. When its content is below the detection limit, the values ​​of C8:0 / DHA and C15:0 / DHA are extremely high. At this time, the minimum value and the identifiable adulteration ratio are calculated with the limit of quantification of DHA as 0.01%FA.

[0040] Table 2-2 Results of the identification of Holstein cow milk that is counterfeit or adulterated with human milk (n=59)

[0041]

[0042] Test results showed that all milk samples were determined to be inconsistent with human milk characteristics, and 1.1% Holstein cow milk could be identified as adulterated in human milk.

[0043] Example 3

[0044] Thirty-two Jersey cow milk samples were taken for verification analysis. The content of characteristic fatty acids in the samples was determined, the corresponding fatty acid ratios were calculated, and compared with the standard human milk fingerprint spectrum constructed in Example 1. If any characteristic fatty acid ratio exceeded the characteristic range, that is, outside the confidence range of the radar chart, the sample was determined to be not completely human milk and to be counterfeit or adulterated. The results are shown in Tables 3-1 and 3-2.

[0045] Table 3-1 Characteristic fatty acid content (% total fatty acids, n=32) of Jersey cow milk adulterated with human milk

[0046]

[0047]

[0048] Note: The DHA content in animal milk is extremely low. When its content is below the detection limit, the values ​​of C8:0 / DHA and C15:0 / DHA are extremely high. At this time, the adulteration ratio can be calculated using the limit of quantification of DHA as 0.01%FA.

[0049] Table 3-2 Identification of the authenticity of Jersey cow milk that is counterfeit or adulterated with human milk (n=32)

[0050]

[0051] Test results showed that all milk samples were determined to be inconsistent with human milk characteristics, and 1.2% Jersey cow milk could be identified as adulterated with human milk.

[0052] Example 4

[0053] Eleven samples of buffalo milk were taken for verification analysis. The content of characteristic fatty acids in the samples was determined, the corresponding fatty acid ratios were calculated, and compared with the standard human milk fingerprint spectrum constructed in Example 1. If any characteristic fatty acid ratio exceeded the characteristic range, that is, outside the confidence range of the radar chart, the sample was determined to be not completely human milk and to be counterfeit or adulterated. The results are shown in Tables 4-1 and 4-2.

[0054] Table 4-1 Characteristic fatty acid content (% total fatty acids, n=11) of buffalo milk adulterated with human milk

[0055]

[0056] Note: The DHA content in animal milk is extremely low. When its content is below the detection limit, the values ​​of C8:0 / DHA and C15:0 / DHA are extremely high. At this time, the adulteration ratio can be calculated using the limit of quantification of DHA as 0.01%FA.

[0057] Table 4-2 Identification of the Authenticity of Buffalo Milk Counterfeited or Adulterated with Human Milk (n=11)

[0058]

[0059]

[0060] Test results showed that all milk samples were determined to be inconsistent with human milk characteristics, and that 1.5% buffalo milk could be identified as adulterated in human milk.

[0061] Example 5

[0062] Twenty-five samples of yak milk were taken for verification analysis. The content of characteristic fatty acids in the samples was determined, the corresponding fatty acid ratios were calculated, and compared with the standard human milk fingerprint spectrum constructed in Example 1. If any characteristic fatty acid ratio exceeded the characteristic range, that is, outside the confidence range of the radar chart, the sample was determined to be not completely human milk and to be counterfeit or adulterated. The results are shown in Tables 5-1 and 5-2.

[0063] Table 5-1 Characteristic fatty acid content (% total fatty acids, n=25) of yak milk adulterated with human milk

[0064]

[0065]

[0066] Note: The DHA content in animal milk is extremely low. When its content is below the detection limit, the values ​​of C8:0 / DHA and C15:0 / DHA are extremely high. At this time, the adulteration ratio can be calculated using the limit of quantification of DHA as 0.01%FA.

[0067] Table 5-2 Identification of Authenticity of Yak Milk Counterfeited or Adulterated with Human Milk (n=25)

[0068]

[0069] Test results showed that all milk samples were determined to be inconsistent with human milk characteristics, and 2.2% yak milk could be identified as adulterated in human milk.

[0070] Example 6

[0071] Thirty-eight samples of goat milk were taken for verification analysis. The content of characteristic fatty acids in the samples was determined, the corresponding fatty acid ratios were calculated, and compared with the standard human milk fingerprint spectrum constructed in Example 1. If any characteristic fatty acid ratio exceeded the characteristic range, that is, outside the confidence range of the radar chart, the sample was determined to be not completely human milk and to be counterfeit or adulterated. The results are shown in Tables 6-1 and 6-2.

[0072] Table 6-1 Characteristic fatty acid content (% total fatty acids, n=38) of goat milk adulterated with human milk

[0073]

[0074]

[0075] Note: The DHA content in animal milk is extremely low. When its content is below the detection limit, the values ​​of C8:0 / DHA and C15:0 / DHA are extremely high. At this time, the adulteration ratio can be calculated using the limit of quantification of DHA as 0.01%FA.

[0076] Table 6-2 Identification of the authenticity of goat milk counterfeited or adulterated with human milk (n=38)

[0077]

[0078] Test results showed that all milk samples were determined to be inconsistent with human milk characteristics, and 2.5% sheep milk could be identified as adulterated in human milk.

[0079] Example 7

[0080] Twenty-seven camel milk samples were taken for verification analysis. The content of characteristic fatty acids in the samples was determined, the corresponding fatty acid ratios were calculated, and the samples were compared with the standard human milk fingerprint spectrum constructed in Example 1. If any characteristic fatty acid ratio exceeded the characteristic range, i.e. outside the confidence range of the radar chart, the sample was determined to be not completely human milk and to be counterfeit or adulterated. The results are shown in Tables 7-1 and 7-2.

[0081] Table 7-1 Characteristic fatty acid content (% total fatty acids, n=27) of camel milk adulterated with human milk

[0082]

[0083]

[0084] Note: The DHA content in animal milk is extremely low. When its content is below the detection limit, the values ​​of C8:0 / DHA and C15:0 / DHA are extremely high. At this time, the adulteration ratio can be calculated using the limit of quantification of DHA as 0.01%FA.

[0085] Table 7-2 Identification of Camel Milk Counterfeit or Adulterated with Human Milk (n=27)

[0086]

[0087] Test results showed that all milk samples were determined to be inconsistent with human milk characteristics, and 4.5% camel milk could be identified as adulterated in human milk.

[0088] Example 8

[0089] Fifteen samples of donkey milk were taken for verification analysis. The content of characteristic fatty acids in the samples was determined, the corresponding fatty acid ratios were calculated, and compared with the standard human milk fingerprint spectrum constructed in Example 1. If any characteristic fatty acid ratio exceeded the characteristic range, that is, outside the confidence range of the radar chart, the sample was determined to be not completely human milk and to be counterfeit or adulterated. The results are shown in Tables 8-1 and 8-2.

[0090] Table 8-1 Characteristic fatty acid content (% total fatty acids, n=15) of donkey milk adulterated with human milk

[0091]

[0092] Note: The DHA content in animal milk is extremely low. When its content is below the detection limit, the values ​​of C8:0 / DHA and C15:0 / DHA are extremely high. At this time, the adulteration ratio can be calculated using the limit of quantification of DHA as 0.01%FA.

[0093] Table 8-2 Identification of the Authenticity of Donkey Milk Counterfeited or Adulterated with Human Milk (n=15)

[0094]

[0095] The test results showed that all milk samples could be determined to be inconsistent with the characteristics of human milk, and that 0.3% human milk could be identified as adulterated in human milk.

[0096] In summary, the method for identifying the authenticity of human milk based on characteristic fatty acid fingerprinting, as described in this paper, was used to verify 59 samples of Holstein cow milk, 32 samples of Jersey cow milk, 11 samples of buffalo milk, 25 samples of yak milk, 38 samples of goat milk, 27 samples of camel milk, and 15 samples of human milk. All 207 other animal milk samples showed characteristic fatty acid ratios exceeding the characteristic range of human milk, with a difference recognition rate of 100%. Furthermore, human milk adulterated with 0.3% to 4.5% or more of other animal milk was effectively identified.

Claims

1. A characteristic fatty acid fingerprint spectrum of human milk, characterized in that... The characteristic fatty acids in human milk are: C8:0, C14:

1. cis-9 C15:0, C15:0 anteiso , C18:2 c9t11, C18:2 c9c12, C20:1 cis-11 C22:6c4c7c10c13c16c19; The ratio range of the characteristic fatty acids is: C8:0 / C18:2 c9c12 0~0.010; C8:0 / C22:6 c4c7c10c13c16c19 0~1.071; C14:1 cis -9 / C20:1 cis -11 0~0.537; C15:0 / C22:6 c4c7c10c13c16c19 0~1.236; C15:0 anteiso / C20:1 cis-11 0~0.333; C18:2 c9t11 / C18:2 c9c12 0~0.

022.

2. A method for identifying the authenticity of human milk using the characteristic fatty acid fingerprint spectrum of human milk as described in claim 1, characterized in that... Includes the following steps: S1: Determine the content of characteristic fatty acids in the test sample and calculate the corresponding ratio of characteristic fatty acids in human milk; S2: Compare the corresponding fatty acid ratios calculated in step S1 with the characteristic fatty acid fingerprint of human milk. If any characteristic fatty acid ratio exceeds the characteristic range, i.e. outside the confidence range of the radar chart, the sample is determined to be not completely human milk and is counterfeit or adulterated.

3. A method for identifying the authenticity of human breast milk as described in claim 2, characterized in that: The determination of the characteristic fatty acid content in the human milk sample to be tested in step S1 is performed by gas chromatography or gas chromatography-mass spectrometry.

4. A method for identifying the authenticity of human breast milk as described in claim 2, characterized in that: The results of calculating the corresponding fatty acid ratios in step S1 are expressed as relative values.

5. A method for identifying the authenticity of human breast milk as described in claim 2, characterized in that: The results of calculating the corresponding fatty acid ratios in step S1 are expressed in absolute values.

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