Determination of compound antioxidants and oxidized flavor substances in milk powder

Abstract

The present invention relates to a compound (natural) antioxidant and a method for determining oxidized flavor substances in milk powder. The compound (natural) antioxidant contains ascorbic acid, ascorbyl palmitate and tocopherol as active ingredients, wherein the mass ratio of the three active ingredients in the compound (natural) antioxidant is: ascorbic acid: ascorbyl palmitate: tocopherol = (1-3.5): (1-7): 1. In the method for determining oxidized flavor substances in milk powder, the compound (natural) antioxidant is used as an oxidative stability improver.

Description

Technical Field

[0001] The invention belongs to the technical field of antioxidants, and in particular relates to a compound antioxidant and a method for determining oxidized flavor substances in milk powder using the compound antioxidant as an oxidative stability improver. Background Art

[0002] The change of milk powder flavor is inseparable from the oxidation of fat, and the fatty acid composition in fat directly affects the lipid oxidation degree of milk powder. Vegetable oil, DHA (docosahexaenoic acid), ARA (arachidonic acid) and other substances are often added to infant formula milk powder to increase the proportion of unsaturated fatty acids, but the more unsaturated fatty acids in milk powder, the easier it is for the product to undergo lipid oxidation reaction; milk lipid oxidation is an extremely complex process, including auto-oxidation, enzymatic oxidation and photosensitive oxidation, among which the most important oxidation reaction of milk powder is auto-oxidation, that is, the direct reaction of milk fat with oxygen molecules, the unsaturated fatty acids in milk fat contact with oxygen in the air or dissolved oxygen in the milk system, and auto-oxidation occurs to generate fat hydroperoxides. Due to the instability of hydroperoxides, there are complex reactions such as decomposition and polymerization of hydroperoxides in the reaction, which produce low molecular weight products with different relative molecular weights and different flavor thresholds and some high molecular weight products, such as aldehydes and ketones, which will affect the quality of milk powder. Aldehydes are chemically active and can be easily oxidized by oxidants or even weak oxidants to form carboxylic acids. Ketones are oxidized to cause the carbonyl group to break from α-C. Although raw milk contains natural antioxidants, most of these substances will be lost or completely removed during processing and storage.

[0003] Milk powder involves a variety of free radicals such as R·, RO·, ROO·, ·OH, etc. The presence of these free radicals will continue to catalyze the oxidation of fat and the generation of aldehydes and ketones, and the oxidation process will accelerate with the increase of temperature. At the same time, the cleavage products will increase, resulting in inaccurate detection results of the content of oxidized flavor substances, reduced test stability, and significant differences in data results (p < 0.05); and riboflavin (VB2) contained in milk powder is a water-soluble photosensitizer, and is currently believed to play an important role in the photooxidation of dairy products and the formation of light-induced odors. After riboflavin is irradiated by natural light, free radicals will be generated and then oxidation reactions will occur. The presence of riboflavin and the photooxidation reaction significantly increase the content of hexanal in the sample, which greatly affects the accuracy of quantitative detection of aldehydes.

[0004] In terms of the pretreatment methods and instrument selection for the detection of flavor substances in milk powder, the separation and identification of volatile components in food at home and abroad often use gas chromatography-mass spectrometry (GC-MS) technology. Gas chromatography-mass spectrometry is one of the most effective tools for identifying volatile and semi-volatile organic compounds. It has high separation performance and accurate identification capabilities. It is often used to detect small molecules that are volatile, low polarity, and low boiling point metabolites, or substances that are volatile after derivatization. It is one of the main instrumental analysis methods at present. Before GC-MS detection, volatile components need to be extracted and enriched. Solid phase microextraction (SPME) is currently the most widely used pretreatment method for flavor substance extraction. SPME performs extraction, enrichment and concentration at the same time. It has the characteristics of mild extraction conditions, high sensitivity, no solvent, and simple operation. For milk powder samples with complex types of volatile flavor substances, low concentrations, small responses, and mild extraction conditions, solid phase microextraction is an ideal and effective pretreatment extraction method.

[0005] Ascorbic acid, also known as vitamin C, is a commonly used natural antioxidant. It uses its ability to be easily oxidized to produce free radicals that are oxidized first, thereby protecting other substances from oxidation. Studies have shown that ascorbic acid can significantly reduce the concentration of oxidized flavor compounds in milk powder; ascorbic acid palmitate is esterified with natural ingredients such as palmitic acid and ascorbic acid. It is a highly effective oxygen scavenger and product enhancer; tocopherol, also known as vitamin E, is one of the most important natural antioxidants and has a strong antioxidant effect.

[0006] At present, most studies are about adding antioxidants to pure oil systems to delay fat oxidation. Although there are some studies that control the oxidation reaction of lipids in milk by applying natural antioxidants to liquid milk systems, the results are often unsatisfactory. In addition, in the process of determining oxidative flavor substances such as aldehydes and ketones, the research on the oxidative stability of milk powder is not sufficient. For example:

[0007] Cited document 1 relates to a quaternary composite antioxidant comprising four natural antioxidants as active ingredients, which specifies the weight ratio of rosemary extract, vitamin C palmitate, vitamin E and tea polyphenol palmitate, and is suitable for various foods, including but not limited to vegetable oil products such as soybean oil and corn oil.

[0008] Reference 2 discloses the addition of catechin (EC) in the production of low-calorie skim milk powder. A portion of skim condensed milk without catechin was used as a blank, and catechin was added to another portion of condensed milk. The results showed that the addition of catechin could reduce the content of oxidized flavor substances in reconstituted milk by 4-8 times.

[0009] Reference 3 applied two natural antioxidants, total organic carbon (TOC) and ascorbic acid (VC), to liquid milk systems. The results showed that the addition of TOC and VC played a significant role in controlling lipid oxidation reactions in milk. Adding ascorbic acid can significantly reduce the concentration of oxidized flavor compounds in milk powder (P<0.05).

[0010] Although the above-mentioned prior art has compounded some natural antioxidants to inhibit the oxidation reaction of lipids in milk, in the determination process of oxidized flavor substances in milk powder, the research on antioxidants that prevent and delay the oxidation process during the experimental determination period is not sufficient, and there is still room for further exploration.

[0011] References:

[0012] Reference 1: CN109022148B

[0013] Cited document 2: Reduction of Stale Flavor Development in Low-Heat Skim MilkPowder via Epicatechin Addition, Journal of Agricultural and Food Chemistry, 2006, 54, 2, 502-508

[0014] Cited document 3: Antioxidant Effect of Tocopherols on Autoxidation of MilkFat, Agricultural and Biological Chemistry, 2014, 878-890 Summary of the invention

[0015] Problem that the invention aims to solve

[0016] As mentioned above, some natural antioxidants related to reducing the oxidation reaction of lipids in milk powder products have been proposed in the art. However, in specific research practice and industrial testing and production, the inventors have also found the following problems:

[0017] The four-component compound antioxidant disclosed in Reference 1 has a synergistic effect with each other and has a better antioxidant capacity than a single antioxidant, but it is mainly targeted at oily substances, such as corn oil, soybean oil, etc. The antioxidant effect of the compound antioxidant in milk powder products is not clear yet.

[0018] Reference 2 studied the effect of catechins (EC) on the formation of off-flavor in low-calorie skim milk powder samples during processing and storage. Although the results showed that catechins played a good antioxidant role as an antioxidant, the study was aimed at skim milk, which has a low fat content and a low degree of lipid oxidation. The actual role of catechins in the changes in milk powder flavor and the high-temperature oxidation of fat was not deeply explored.

[0019] The study cited in reference 3 showed that total organic carbon (TOC) and ascorbic acid (V C ) These two antioxidants can inhibit the oxidation reaction of lipids, but the results show that the concentrations of hexanal, heptanal, 2-heptanone, 2-nonanone and 3-octen-2-one in the milk powder with added TOC showed a slightly increasing trend. The ESR spectrum data analysis showed that the peak morphology of the milk powder with added TOC and the blank was similar, which indicates that the antioxidant effect of total organic carbon (TOC) on milk powder may not be obvious.

[0020] The above-mentioned prior art does not disclose a more effective compound antioxidant for preventing and delaying the oxidation process when determining the oxidized flavor substances in milk powder, and does not explore the most appropriate addition scheme to ensure more accurate qualitative and quantitative determination of the content of oxidized flavor substances in milk powder during the shelf life.

[0021] Furthermore, it is known that in the SPME extraction process, most analytical methods are to place the milk powder sample at a temperature of 60°C to 80°C for heating and shaking equilibrium for 30min to 50min, then insert the solid phase extraction head into the headspace extraction bottle for extraction and adsorption for 30min to 50min, and finally analyze by GC-MS for 40min to 50min. It can be seen from the shortcomings of the prior art that the sample is in a high temperature state and heated for a long time, which will accelerate the oxidation reaction process, increase the cleavage products, and cause the measured content of oxidized flavor substances to fluctuate within a certain range, resulting in differences in the results.

[0022] Therefore, in view of the above-mentioned deficiencies in the prior art, the problem to be solved by the present invention is to provide a compound antioxidant, especially a compound natural antioxidant, which can prevent and delay the oxidation process in the determination of oxidized flavor substances in milk powder, so as to slow down or eliminate the oxidation reaction in the detection process, ensure the stability of the results of the instrumental determination of the oxidized flavor substances in the milk powder and the parallelism of the data, so as to more accurately qualitatively and quantitatively determine the content of oxidized flavor substances in the milk powder during the shelf life.

[0023] Solutions for solving problems

[0024] Through long-term research by the inventors of the present invention, it is found that the above technical problems can be solved by the following technical solutions:

[0025] [1]. The present invention first provides a composite (natural) antioxidant for assisting the detection of flavor substances in milk powder products, wherein:

[0026] The compound (natural) antioxidant contains ascorbic acid, ascorbic acid ester and tocopherol as active ingredients,

[0027] The mass ratio of the three active ingredients in the compound (natural) antioxidant is ascorbic acid: ascorbic acid ester: tocopherol = (1-3.5): (1-7): 1.

[0028] [2] The compound (natural) antioxidant according to [1], wherein the mass ratio of ascorbic acid, ascorbic acid ester and tocopherol in the compound (natural) antioxidant is: ascorbic acid: ascorbyl palmitate: tocopherol = (2.5-3.5): (3-4): 1.

[0029] [3] The compound (natural) antioxidant according to [1] or [2], wherein the amount of the compound (natural) antioxidant added is 0.01% to 0.1% by mass based on the total mass of the milk powder product.

[0030] [4] The compound (natural) antioxidant according to [1] or [2], wherein, based on the total mass of the milk powder product, the contents of ascorbic acid, ascorbic acid ester and tocopherol in the compound (natural) antioxidant are 0.0036% to 0.03% by mass, 0.0036% to 0.061% by mass and 0.0036% to 0.0087% by mass respectively.

[0031] [5]. The composite (natural) antioxidant according to any one of [1] to [4], wherein the ascorbic acid is selected from one or more of plant extracts and artificial chemical synthesis; the ascorbic acid ester is selected from one or more of artificial chemical synthesis; and the tocopherol is selected from one or more of artificial chemical synthesis and artificial extracts.

[0032] [6] The composite (natural) antioxidant according to any one of [1] to [5], wherein the ascorbic acid ester is selected from saturated fatty acid esters of ascorbic acid having 12 to 20 carbon atoms.

[0033] [7]. The compound (natural) antioxidant according to any one of [1] to [6], wherein the milk powder products include: powdered, solid, semi-solid and liquid milk powder products.

[0034] [8]. The present invention further provides a method for testing oxidized flavor substances in milk powder products, characterized in that the testing method comprises:

[0035] Pre-processing steps,

[0036] The extraction steps,

[0037] The steps of detection,

[0038] Wherein, in the pre-treatment step, the compound (natural) antioxidant described in any one of [1] to [7] above is added to the milk powder product to be tested to obtain a test sample;

[0039] In the extraction step, the test sample is extracted by a solid phase extraction method to collect flavor substances.

[0040] [9] The testing method according to [8], wherein the detection step includes using chromatography and mass spectrometry to detect the flavor substances collected in the extraction step.

[0041]

[10] . In addition, the present invention also provides an application of the composite antioxidant described in any one of [1] to [7] or the test method for oxidized flavor substances in milk powder products described in [8] or [9] in the field of sensory evaluation of milk powder.

[0042] Effects of the Invention

[0043] Through the implementation of the above technical solution, the present invention has the following advantages and can obtain the following technical effects:

[0044] 1) The compound (natural) antioxidant provided by the present invention can effectively prevent or delay the oxidation process caused by long-term high-temperature heating, oscillation extraction, light induction, etc. of the sample during the determination of milk powder samples by optimizing the proportion of known (natural) antioxidants.

[0045] 2) The method for determining oxidized flavor substances in milk powder provided by the present invention can ensure the material stability and data parallelism of the instrumental determination results, and can more accurately determine and more reasonably reflect the content of oxidized flavor substances in milk powder during the shelf life.

[0046] 3) The method for determining flavor substances in milk powder provided by the present invention is preferably a method for improving the oxidative stability of major oxidized flavor substances such as aldehydes and ketones in milk powder products during instrument detection, which can improve the oxidative stability of major oxidized flavor substances in milk powder and the data parallelism of the determination results. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Description of Reference Numerals

[0048] Figure 1:Effects of three natural antioxidants on the contents of main oxidized flavor substances in milk powder in a single factor experiment

[0049] Figure 2 :Effects of different compounding forms of antioxidants on the contents of main oxidized flavor substances in milk powder in a pairwise compounding experiment

[0050] Figure 3 :Total ion flow chart of blank milk powder sample (without adding any antioxidant)

[0051] Figure 4 :Total ion current diagram of instrument detection results of milk powder sample in Experimental Example 14 DETAILED DESCRIPTION

[0052] The following is a detailed description of the present invention. The following description of the technical features is based on representative embodiments and specific examples of the present invention, but the present invention is not limited to these embodiments and specific examples. It should be noted that:

[0053] In this specification, the numerical range expressed using "a numerical value A to a numerical value B" means a range including the endpoints numerical values ​​A and B.

[0054] In the present specification, a numerical range expressed using "above" or "below" means a numerical range including the number.

[0055] In this specification, the word "may" means both performing a certain process and not performing a certain process.

[0056] In this specification, the use of "optional" or "optional" indicates that certain substances, components, execution steps, application conditions and other factors are used or not used.

[0057] In this specification, the "room temperature" used means an indoor ambient temperature of "20°C".

[0058] In this specification, the unit names used are all international standard unit names, and unless otherwise stated, the "%" used indicates weight or mass percentage.

[0059] In this specification, the use of "substantially" means that the standard deviation from a theoretical model or theoretical data is within a range of 3%, preferably 2%, and more preferably 1%.

[0060] In this specification, the references to "some specific / preferred embodiments", "other specific / preferred embodiments", "embodiments", etc., mean that the specific elements (e.g., features, structures, properties and / or characteristics) described in connection with the embodiments are included in at least one embodiment described herein, and may or may not exist in other embodiments. In addition, it should be understood that the elements may be combined in various embodiments in any suitable manner.

[0061] <First aspect>

[0062] In the first aspect of the present invention, a composite antioxidant, in particular a composite natural antioxidant, is provided for use in the determination of oxidized flavor substances in milk powder products. The composite (natural) antioxidant contains ascorbic acid, ascorbic acid esters and tocopherol as active ingredients, and the three antioxidants have an obvious synergistic effect, which can reduce the oxidation process of milk powder caused by long-term high-temperature heating, oscillation extraction, light induction and other reasons in the process of determining oxidized flavor substances in milk powder products, so that the determination result is more accurate.

[0063] The technical solution of the present invention is mainly obtained based on the following insights:

[0064] The present invention utilizes solid phase microextraction and chromatography-mass spectrometry to analyze the contents of oxidized flavor substances such as hexanal, nonanal, 2-heptanone, etc. in milk powder, and obtains the optimal synergistic compounding ratio of the three antioxidants through single factor experiments, two-by-two compounding, and compounding of different types and doses of (natural) antioxidants ascorbic acid, ascorbic acid esters, and tocopherol, thereby effectively improving the oxidative stability of the main oxidized flavor substances in the milk powder, the data parallelism, and the accuracy of the measurement results.

[0065] In addition, based on the above insights, the present invention also provides a compound (natural) antioxidant that can be used to stabilize oxidation during storage of milk powder products.

[0066] <Dairy powder products>

[0067] In the present invention, the milk powder products include products based on or mainly based on human milk or animal milk components. There is no particular limitation on animal milk, which can generally include dairy products of cow's milk, goat's milk, and camel's milk, preferably, dairy products of cow's milk and / or goat's milk.

[0068] In principle, the present invention has no special requirements for the form of dairy products, for example, it can be a powdered dairy product, a solid block dairy product, a semi-solid dairy product or a liquid dairy product. In some preferred embodiments of the present invention, the above-mentioned dairy product of the present invention can be a powdered dairy product. For example, it can be a milk powder type dairy product for infants, children or middle-aged and elderly people. In the present invention, the infant refers to an infant aged 0 to 3 years old, and the child refers to a child older than 3 years old and younger than 12 years old.

[0069] For the above-mentioned dairy products, in addition to animal milk, one or more of the following ingredients may be added as needed: edible oil products, fermented dairy products, protein-added components, vitamin-added components, probiotic-added components, etc.

[0070] There is no particular limitation on the use of oil products in the present invention. For example, it may refer to a single product or a mixture of animal or vegetable oils processed by one or more methods of refining, hydrogenation, transesterification, and fractionation, with (or without) the addition of water and other auxiliary materials, and a solid, semi-solid or flowing oil product with certain properties manufactured by (or without) emulsification, rapid cooling and kneading.

[0071] Fermented dairy products are not particularly limited in the present invention, for example, they may be cheese that has been (semi) fermented, liquid or semi-solid fermented milk, etc. In the present invention, according to the provisions of GB 5420-2021, "cheese" is a mature or immature soft, semi-hard, hard or extra-hard dairy product that may be coated, wherein the ratio of whey protein / casein does not exceed the corresponding ratio in cow (or other dairy livestock) milk (except whey cheese). The cheese is obtained by any of the following methods: a) after the protein in milk and / or (or) dairy products is coagulated or partially coagulated under the action of rennet or other appropriate coagulants (or directly using the curd after coagulation as raw material), adding or not adding fermentation bacteria, edible salt, food additives, food nutrient fortifiers, discharging or not discharging whey (when the protein curd after coagulation is used as raw material), and fermenting or not fermenting to obtain a solid or semi-solid product; b) the processing technology includes the coagulation process of protein in milk and (or) dairy products, and gives the finished product similar physical, chemical and sensory properties to the product described in a). In the present invention, according to the provisions of GB 19302-2010, "fermented milk" refers to a product with a reduced pH value made from raw bovine (sheep) milk or milk powder after sterilization and fermentation; wherein the fat is greater than or equal to 3.1g / 100g, the non-fat milk solids are greater than or equal to 8.1g / 100g, the protein is greater than or equal to 2.9g / 100g, and the acidity is greater than or equal to 70.0°T.

[0072] The present invention has no particular limitation on the protein additive component, and the protein additive component may be, for example, various protein concentrates or additives derived from plants, animals, or artificial synthesis.

[0073] The compound natural antioxidant provided by the invention is particularly suitable for detecting oxidized flavor substances in infant formula milk powder products.

[0074] <Complex Antioxidant>

[0075] As mentioned above, the compound antioxidant of the present invention contains ascorbic acid, ascorbic acid ester and tocopherol as active ingredients, and by optimizing the usage amount, a synergistic antioxidant stabilization effect can be obtained. The raw materials of the compound antioxidant can mainly come from natural ingredients. In a preferred embodiment of the present invention, the compound antioxidant is also a compound natural antioxidant.

[0076] (ascorbic acid)

[0077] In the present invention, ascorbic acid is used as one of the essential components. The type and source of ascorbic acid are not particularly limited in principle. Preferably, L-ascorbic acid can be used.

[0078] Ascorbic acid, also known as vitamin C, is a naturally occurring, water-soluble vitamin with antioxidant properties. Pure ascorbic acid is a white solid, but some samples with impurities may have a slightly yellowish tint. Ascorbic acid is a six-carbon lactone produced by plants and some animals, but not by humans and other primates. It occurs naturally in many fresh fruits and vegetables, from oranges and grapefruits to broccoli, kale, and tomatoes. However, when in these foods, vitamin C may be reduced by heat, boiling water, or air. Ascorbic acid functions as an enzymatic cofactor for a variety of enzymes, serving as an electron donor for monooxygenases and dioxygenases. Ascorbic acid is also a powerful antioxidant, especially to reactive oxygen species. As an antioxidant, ascorbic acid helps prevent cell damage caused by free radicals (unstable molecules that can damage cells). In addition, ascorbic acid is a Generally Recognized As Safe (GRAS) substance and can be used as a chemical preservative in foods and as a nutritional or dietary supplement.

[0079] The source of ascorbic acid in the present invention is not particularly limited, and it can be extracted from fruits and vegetables, or obtained by industrial synthesis via glucose. In other specific embodiments, the ascorbic acid used in the present invention can also be obtained by commercial purchase.

[0080] Furthermore, in the present invention, there is no particular limitation on the use of L-ascorbic acid. Preferably, it can be prepared as an aqueous solution for use.

[0081] (Ascorbyl ester)

[0082] As for the ascorbic acid ester of the present invention, there is no particular limitation in principle as long as the above-mentioned technical effects of the present invention can be achieved. In some preferred embodiments of the present invention, the ascorbic acid ester can generally be a saturated fatty acid ester of ascorbic acid with a carbon number of 12 to 20. In a further preferred embodiment of the present invention, the ascorbic acid ester of the present invention includes palmitic acid ester of ascorbic acid.

[0083] Ascorbyl palmitate is a highly effective, safe, non-toxic, fat-soluble nutritional antioxidant that is insoluble in water and vegetable oil. It is a white or yellowish-white powder with a slight citrus aroma.

[0084] Ascorbyl palmitate is a vitamin C derivative formed by the esterification of natural ingredients ascorbic acid and palmitic acid. It not only enhances the stability of vitamin C, but also retains its physiological value. It is non-toxic to the human body and is resistant to high temperatures. It is the only antioxidant permitted by my country's "Food Additives Hygiene Standards" for use in infant foods. It can scavenge free radicals and, at the same time, in the presence of pro-oxidant metal ions such as Cu 2+ and Fe 3+ Under the action of the like, ascorbyl palmitate can react with oxygen to form dehydroascorbyl palmitate, thereby removing oxygen from the oil and delaying the generation of oxidation reactions.

[0085] The source of ascorbyl palmitate is not particularly limited in the present invention, and can be artificially synthesized by methods such as enzyme exchange reaction, acyl halide esterification and enzyme catalysis, or can be esterified by natural ingredients such as palmitic acid and L-ascorbic acid. Further, the use of ascorbyl palmitate is not particularly limited in the present invention and can be used in the form of a solution, emulsion or suspension. In some preferred embodiments, it can be prepared as a solution such as ethanol, fatty acid (ester) and used.

[0086] (Tocopherol)

[0087] In the present invention, tocopherol is used as one of the essential components. The type of tocopherol is not particularly limited in principle, and preferably, α-tocopherol can be used.

[0088] Tocopherol is the active form of vitamin E, a yellow to reddish brown clear viscous liquid. Natural tocopherols are all D-tocopherol (dextral type), which has 8 isomers such as α, β, γ, δ, among which α-tocopherol has the strongest activity. Tocopherol is fat-soluble due to its long side chain, and can directly remove free radicals by breaking the O-C bond on the tocopherol free radical oxa-naphthalene ring and combining ·OH. It is widely used in whole milk powder, cream or margarine, meat products, aquatic processed products, dehydrated vegetables, fruit juice drinks, frozen foods and instant foods, etc., especially tocopherol is more important as an antioxidant and nutritional fortifier for baby food, therapeutic food, fortified food, etc.

[0089] The source of tocopherol is not particularly limited in the present invention, and can be derived from artificial chemical synthesis, or can be derived from various natural tocopherol artificial extracts. Further, the use mode of tocopherol is not particularly limited in the present invention, and can be used in the form of solution, emulsion or suspension. In some preferred embodiments, it can be prepared as solutions such as ethanol, fatty acid (ester) and used.

[0090] (Compound)

[0091] In the prior art, ascorbic acid, ascorbic acid esters and tocopherol are all commonly used antioxidants in the food processing industry. The present invention discovers and reports for the first time that when the three are reasonably configured, they show a synergistic effect on inhibiting the (high temperature) oxidation of milk powder products. At the same time, in the detection of dairy products, it is also found that when the three are reasonably configured, the oxidation of milk powder caused by high temperature heat experience can be avoided, thereby making the test results closer to the actual situation.

[0092] Specifically, the present invention firstly conducts single factor experiments on the addition amounts of three antioxidants respectively to study the influence of ascorbic acid, ascorbic acid ester and tocopherol on the antioxidant effect of oxidized flavor substances in milk powder; then the three natural antioxidants are compounded in pairs to obtain their antioxidant effects on the oxidized flavor substances in milk powder; finally, the three natural antioxidants are compounded at the same time to obtain the best combination ratio so that the three natural antioxidants can exert the best antioxidant effect.

[0093] Specifically, in some specific embodiments of the present invention, the mass ratio of ascorbic acid, ascorbic acid ester and tocopherol can be (1-3.5):(1-7):1, preferably (2-3.5):(1-6):1, more preferably (2.5-3.5):(2-6):1, and most preferably (2.5-3.5):(3-4):1.

[0094] In addition, as long as the above-mentioned usage ratio is met, the dosage of ascorbic acid, ascorbic acid esters, and tocopherol (especially the maximum dosage or the total dosage of the three) in the compound natural antioxidant provided by the present invention is not limited in principle, and the dosage of each of the three and the total dosage standard can be limited in accordance with different types of milk powder products in accordance with the Food Safety Law or relevant management regulations.

[0095] In some specific embodiments of the present invention, from the perspective of saving materials and measuring efficiency, in some specific embodiments, from the perspective of ensuring antioxidant effect, based on the total mass of milk powder, the content of ascorbic acid can be 0.0036% to 0.03% by mass, preferably 0.004% to 0.025% by mass, more preferably 0.008% to 0.02% by mass; the content of ascorbic acid ester can be 0.0036% to 0.061% by mass, preferably 0.005% to 0.05% by mass, more preferably 0.009% to 0.04%; the content of tocopherol can be 0.0036% to 0.0087% by mass, preferably 0.0033% to 0.008%, more preferably 0.0041% to 0.005%. If the content is too low, there is concern that the antioxidant effect is not good in some cases, and if the content is too high, it will also cause waste of materials or safety risks. In addition, the total amount of the compound antioxidant described in the present invention can be 0.01% to 0.1% by mass, preferably 0.02% to 0.08% by mass.

[0096] In addition, for the composite antioxidant of the present invention, in addition to comprising the above-mentioned three necessary components, optionally according to any other needs, other antioxidant components may also be included. The present invention is not particularly limited to the type of other antioxidant components, and may be natural or synthetic antioxidant components commonly used in the food field. In addition, in some specific embodiments of the present invention, for the content of these other antioxidant components, the total mass of the antioxidant components, the content of other antioxidant components is below 30 mass %, preferably below 20 mass %, more preferably below 10 mass %, and in some other embodiments, the content of the above-mentioned other antioxidant components is substantially zero.

[0097] <Second Aspect>

[0098] In the second aspect of the present invention, there is provided a method for using the compound antioxidant provided in the first aspect.

[0099] In some preferred embodiments of the present invention, the compound natural antioxidant of the present invention can be used in the detection of oxidized flavor substances in milk powder products to avoid further oxidation of the test object due to the thermal experience of heating from room temperature to high temperature such as extraction temperature during the detection, thereby causing deviations in the test results. In particular, the compound natural antioxidant of the present invention is very suitable for the detection of flavor substances in human milk or infant formula milk products, and has improved or enhanced test results.

[0100] The above detection may generally include extraction steps and chromatography-mass spectrometry detection steps.

[0101] In some specific embodiments, the extraction step can generally use a solid phase extraction-headspace enrichment method to collect flavor / odor related substances in the test object. The present invention has no particular limitations on the solid phase extraction and headspace enrichment methods, and they can be performed using conventional methods and equipment in the art.

[0102] For chromatography-mass spectrometry detection, in some specific embodiments, the positions obtained by the headspace enrichment method can usually be subjected to gas chromatography-mass spectrometry detection to analyze the types and contents of various flavor substances. In addition, for the quantitative analysis method, preferably, the internal standard method can be used for quantitative analysis.

[0103] Furthermore, the present invention has no particular limitation on the manner of combining chromatography and mass spectrometry, and any combination or operation manner known or existing in the art for chromatography-mass spectrometry odorant detection may be adopted.

[0104] The odor substances described in the present invention are not particularly limited, and generally may include ketone substances, aldehyde substances, etc. In some specific implementation schemes, the specifications of the detection instrument and the provisions in the detection standard are relevant.

[0105] In the past, in the detection of odor substances, especially when enriching odor substances by (solid phase) extraction-desorption, it is necessary to heat to a high temperature and keep it for a certain period of time to collect the odor position. At this time, observable oxidation reactions still occur in the detection object. Therefore, there is a certain deviation between the flavor substances finally measured and the actual content of flavor substances in the detection object.

[0106] In the present invention, the above-mentioned problem can be solved by a pre-treatment step, that is, the detection object (such as milk powder products) is mixed with the compound natural antioxidant provided by the present invention to obtain the detection object after pre-treatment.

[0107] Furthermore, the detection sample can be prepared by the above extraction-enrichment method, and then the flavor substances therein can be detected by chromatography-mass spectrometry.

[0108] Example

[0109] The present invention will be further described in detail below through specific examples. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0110] 1.1 Raw materials

[0111] In the following Examples section, unless otherwise stated, the starting materials used were as follows.

[0112] Infant formula milk powder: Heilongjiang Feihe Dairy Co., Ltd.

[0113] L-ascorbic acid: Merck Sigma Aldrich

[0114] Ascorbyl palmitate: Merck Sigma Aldrich

[0115] α-Tocopherol: Merck Sigma Aldrich

[0116] In the following sections, unless otherwise stated, when referring to the mass parts and ratios of the ingredients in the complex natural antioxidants, the mass parts refer to the mass parts of L-ascorbic acid, ascorbyl palmitate, and α-tocopherol as active ingredients, and the ratio refers to the mass ratio of L-ascorbic acid, ascorbyl palmitate, and α-tocopherol as active ingredients.

[0117] 1.2 Main Reagents

[0118] 2-Methyl-3-heptanone internal standard (Merck Sigma Aldrich), sodium chloride (analytical grade), etc.

[0119] 1.3 Experimental methods

[0120] 1.3.1 Pre-processing steps

[0121] Weigh 2.00g-3.00g of milk powder, add 10mL-15mL of pure water into a 50mL centrifuge tube, pour in the milk powder, shake evenly, take 8mL-10mL of sample and 1g of sodium chloride into a 20mL headspace bottle, add 15μL-20μL of 2-methyl-3-heptanone internal standard (1000μg / mL), and tighten the bottle cap.

[0122] 1.3.2 Automated solid phase microextraction

[0123] A 80 μm DVB / CWR / PDMS extraction head was selected, balanced heating was performed at 65°C for 20 min-40 min, extraction was performed for 30 min-40 min, and desorption was performed at 250°C for 3-6 min.

[0124] 1.3.3 GC-MS analysis

[0125] Gas phase parameters: HP-5MS quartz capillary column, split ratio 0-10:1; injection port temperature 220℃-250℃, initial temperature 40℃ maintained for 3min, heated to 170℃ at 5℃ / min and maintained for 5min, heated to 240℃ at 20℃ / min and maintained for 10min; carrier gas was He, column flow rate 1mL / min.

[0126] Mass spectrometry parameters: ionization mode: EI source, ionization energy: 70 eV, ion source temperature: 150 °C, interface temperature: 250 °C; full scan mode, scanning mass range: 40-400 m / z.

[0127] 1.4 Statistical analysis

[0128] The experimental data were expressed as mean ± standard deviation (mean ± SD). Each experiment was repeated at least three times. GraphPad Prism 8.00, Excel 2018 and R language were used to draw charts. SPSS 16.0 software was used for statistical analysis of the experimental data. One-way analysis of variance (Duncan's multiple range test) was used. P < 0.05 indicated a significant difference.

[0129] <Single factor experimental comparison>

[0130] Comparative Example 1

[0131] Experimental method: Weigh 2.00g milk powder, add 11mL pure water to a 50mL centrifuge tube, pour in the milk powder, shake evenly, take 8mL sample and 1g sodium chloride and put them into a 20mL headspace bottle, add 16μL 2-methyl-3-heptanone internal standard (1000μg / mL), add L-ascorbic acid, ascorbyl palmitate and α-tocopherol to the milk powder sample at an addition amount of 0.03% (calculated based on the mass of milk powder), screw on the bottle cap, and use solid phase microextraction and gas chromatography-mass spectrometry to determine the content of oxidized flavor substances (hexanal, nonanal, 2-heptanone) in the sample (refer to 1.3.2~1.3.3 for parameters), and perform data analysis.

[0132] Table 1: Content of oxidized flavor substances in milk powder samples with added L-ascorbic acid, ascorbyl palmitate, and α-tocopherol

[0133]

[0134] Note: a / b / c marked in the upper right corner of the number: the same letters indicate no significant difference (P>0.05), and different letters indicate significant difference (P<0.05)

[0135] From Table 1 and Figure 1The results showed that the total content of flavor substances in the blank control group without adding antioxidants was 47.80μg / kg; the oxidative stability effects of the three natural antioxidants were: L-ascorbic acid (35.52μg / kg) > ascorbyl palmitate (35.96μg / kg) > α-tocopherol (36.88μg / kg), but there was no significant difference in the total content of flavor substances obtained by adding the three natural antioxidants (P>0.05). Therefore, it can be concluded from the single-factor experiment that the three natural antioxidants can all have an oxidative stability effect on oxidized flavor substances in milk powder, and the effects are similar, among which L-ascorbic acid has the best oxidative stability effect.

[0136] <Comparative Example of Two-by-Two Compounding Experiment>

[0137] Comparative Example 2

[0138] Experimental method: Weigh 2.00g milk powder, add 11mL pure water to a 50mL centrifuge tube, pour in the milk powder, shake evenly, take 8mL sample and 1g sodium chloride and put them into a 20mL headspace bottle, add 16μL 2-methyl-3-heptanone internal standard (1000μg / mL), add L-ascorbic acid, ascorbyl palmitate, and α-tocopherol at an addition amount of 0.03% (calculated based on the mass of milk powder), and add them to the milk powder sample in pairs. The compounding scheme includes: L-ascorbic acid: ascorbyl palmitate (1:1), L-ascorbic acid: α-tocopherol (1:1), ascorbyl palmitate: α-tocopherol (1:1), tighten the bottle cap, and use solid phase microextraction and gas chromatography-mass spectrometry to determine the content of oxidized flavor substances (hexanal, nonanal, 2-heptanone) in the sample (refer to 1.3.2~1.3.3 for parameters), and perform data analysis.

[0139] Table 2: Content of oxidized flavor substances in milk powder samples with three antioxidants added in pairs

[0140]

[0141] Note: a / b / c marked in the upper right corner of the numbers: the same letters indicate no significant difference (P>0.05), and different letters indicate a significant difference (P<0.05).

[0142] From Table 2 and Figure 2The results showed that the oxidation stability effect of the three natural antioxidants in combination was as follows: L-ascorbic acid: ascorbyl palmitate (32.67 μg / kg) > ascorbyl palmitate: α-tocopherol (33.69 μg / kg) > L-ascorbic acid: α-tocopherol (34.93 μg / kg); there was a significant difference between the two groups of compound antioxidants without ascorbyl palmitate and the compound antioxidants with ascorbyl palmitate (P < 0.05). From the experiment of the three natural antioxidants in combination, it can be concluded that the oxidation stability effect of the three natural antioxidants in combination is better than the oxidation stability effect of the three antioxidants added alone, among which the compound antioxidant (L-ascorbic acid: ascorbyl palmitate) has the best effect.

[0143] <Combination Experiment Example>

[0144] Experimental Example 1:

[0145] When the total amount of the three antioxidants was 0.03% (calculated by the mass of milk powder), the compounding ratio of the three antioxidants was set to L-ascorbic acid: ascorbyl palmitate: α-tocopherol = 1:1:1, and the influence on the stability of the content of oxidized flavor substances in milk powder was detected by instrument at 65°C. The mean value of the total content of oxidized flavor substances (hexanal, nonanal, 2-heptanone) was used as the evaluation index. The results are shown in Table 3.

[0146] Experimental Example 2:

[0147] When the total amount of the three antioxidants was 0.03% (calculated by the mass of milk powder), the compounding ratio of the three antioxidants was set to L-ascorbic acid: ascorbyl palmitate: α-tocopherol = 1:3:1, and the influence on the stability of the content of oxidized flavor substances in milk powder was detected by instrument at 65°C. The mean value of the total content of oxidized flavor substances (hexanal, nonanal, 2-heptanone) was used as the evaluation index. The results are shown in Table 3.

[0148] Experimental Example 3:

[0149] When the total amount of the three antioxidants was 0.03% (calculated by the mass of milk powder), the compounding ratio of the three antioxidants was set to L-ascorbic acid: ascorbyl palmitate: α-tocopherol = 1:5:1, and the influence on the stability of the content of oxidized flavor substances in milk powder was detected by instrument at 65°C. The mean value of the total content of oxidized flavor substances (hexanal, nonanal, 2-heptanone) was used as the evaluation index. The results are shown in Table 3.

[0150] Experimental Example 4:

[0151] When the total amount of the three antioxidants was 0.03% (calculated by the mass of milk powder), the compounding ratio of the three antioxidants was set to L-ascorbic acid: ascorbyl palmitate: α-tocopherol = 1:7:1, and the influence on the stability of the content of oxidized flavor substances in milk powder was detected by instrument at 65°C. The mean value of the total content of oxidized flavor substances (hexanal, nonanal, 2-heptanone) was used as the evaluation index. The results are shown in Table 3.

[0152] Experimental Example 5:

[0153] When the total amount of the three antioxidants was 0.03% (calculated by the mass of milk powder), the compounding ratio of the three antioxidants was set to L-ascorbic acid: ascorbyl palmitate: α-tocopherol = 2:1:1, and the influence on the stability of the content of oxidized flavor substances in milk powder was detected by instrument at 65°C. The mean value of the total content of oxidized flavor substances (hexanal, nonanal, 2-heptanone) was used as the evaluation index. The results are shown in Table 3.

[0154] Experimental Example 6:

[0155] When the total amount of the three antioxidants was 0.03% (calculated by the mass of milk powder), the compounding ratio of the three antioxidants was set to L-ascorbic acid: ascorbyl palmitate: α-tocopherol = 2:3:1, and the influence on the stability of the content of oxidized flavor substances in milk powder was detected by instrument at 65°C. The mean value of the total content of oxidized flavor substances (hexanal, nonanal, 2-heptanone) was used as the evaluation index. The results are shown in Table 3.

[0156] Experimental Example 7:

[0157] When the total amount of the three antioxidants was 0.03% (calculated by the mass of milk powder), the compounding ratio of the three antioxidants was set to L-ascorbic acid: ascorbyl palmitate: α-tocopherol = 2:5:1, and the influence on the stability of the content of oxidized flavor substances in milk powder was detected by instrument at 65°C. The mean value of the total content of oxidized flavor substances (hexanal, nonanal, 2-heptanone) was used as the evaluation index. The results are shown in Table 3.

[0158] Experimental Example 8:

[0159] When the total amount of the three antioxidants was 0.03% (calculated by the mass of milk powder), the compounding ratio of the three antioxidants was set to L-ascorbic acid: ascorbyl palmitate: α-tocopherol = 2:7:1, and the influence on the stability of the content of oxidized flavor substances in milk powder was detected by instrument at 65°C. The mean value of the total content of oxidized flavor substances (hexanal, nonanal, 2-heptanone) was used as the evaluation index. The results are shown in Table 3.

[0160] Experimental Example 9:

[0161] When the total amount of the three antioxidants was 0.03% (calculated by the mass of milk powder), the compounding ratio of the three antioxidants was set to L-ascorbic acid: ascorbyl palmitate: α-tocopherol = 2.5:1:1, and the influence on the stability of the content of oxidized flavor substances in milk powder was detected by instrument at 65°C. The mean value of the total content of oxidized flavor substances (hexanal, nonanal, 2-heptanone) was used as the evaluation index. The results are shown in Table 3.

[0162] Experimental Example 10:

[0163] When the total amount of the three antioxidants was 0.03% (calculated by the mass of milk powder), the compounding ratio of the three antioxidants was set to L-ascorbic acid: ascorbyl palmitate: α-tocopherol = 2.5:3:1, and the influence on the stability of the content of oxidized flavor substances in milk powder was detected by instrument at 65°C. The mean value of the total content of oxidized flavor substances (hexanal, nonanal, 2-heptanone) was used as the evaluation index. The results are shown in Table 3.

[0164] Experimental Example 11:

[0165] When the total amount of the three antioxidants was 0.03% (calculated by the mass of milk powder), the compounding ratio of the three antioxidants was set to L-ascorbic acid: ascorbyl palmitate: α-tocopherol = 2.5:5:1, and the influence on the stability of the content of oxidized flavor substances in milk powder was detected by instrument at 65°C. The mean value of the total content of oxidized flavor substances (hexanal, nonanal, 2-heptanone) was used as the evaluation index. The results are shown in Table 3.

[0166] Experimental Example 12:

[0167] When the total amount of the three antioxidants was 0.03% (calculated by the mass of milk powder), the compounding ratio of the three antioxidants was set to L-ascorbic acid: ascorbyl palmitate: α-tocopherol = 2.5:7:1, and the influence on the stability of the content of oxidized flavor substances in milk powder was detected by instrument at 65°C. The mean value of the total content of oxidized flavor substances (hexanal, nonanal, 2-heptanone) was used as the evaluation index. The results are shown in Table 3.

[0168] Experimental Example 13:

[0169] When the total amount of the three antioxidants was 0.03% (calculated by the mass of milk powder), the compounding ratio of the three antioxidants was set to L-ascorbic acid: ascorbyl palmitate: α-tocopherol = 3.5:1:1, and the influence on the stability of the content of oxidized flavor substances in milk powder was detected by instrument at 65°C. The mean value of the total content of oxidized flavor substances (hexanal, nonanal, 2-heptanone) was used as the evaluation index. The results are shown in Table 3.

[0170] Experimental Example 14:

[0171] When the total amount of the three antioxidants was 0.03% (calculated by the mass of milk powder), the compounding ratio of the three antioxidants was set to L-ascorbic acid: ascorbyl palmitate: α-tocopherol = 3.5:3:1, and the influence on the stability of the content of oxidized flavor substances in milk powder was detected by instrument at 65°C. The mean value of the total content of oxidized flavor substances (hexanal, nonanal, 2-heptanone) was used as the evaluation index. The results are shown in Table 3.

[0172] Experimental Example 15:

[0173] When the total amount of the three antioxidants was 0.03% (calculated by the mass of milk powder), the compounding ratio of the three antioxidants was set to L-ascorbic acid: ascorbyl palmitate: α-tocopherol = 3.5:5:1, and the influence on the stability of the content of oxidized flavor substances in milk powder was detected by instrument at 65°C. The mean value of the total content of oxidized flavor substances (hexanal, nonanal, 2-heptanone) was used as the evaluation index. The results are shown in Table 3.

[0174] Experimental Example 16:

[0175] When the total amount of the three antioxidants was 0.03% (calculated by the mass of milk powder), the compounding ratio of the three antioxidants was set to L-ascorbic acid: ascorbyl palmitate: α-tocopherol = 3.5:7:1, and the influence on the stability of the content of oxidized flavor substances in milk powder was detected by instrument at 65°C. The mean value of the total content of oxidized flavor substances (hexanal, nonanal, 2-heptanone) was used as the evaluation index. The results are shown in Table 3.

[0176] Table 3: Effect of the compounding ratio of three antioxidants on the content of oxidized flavor substances in milk powder samples

[0177]

[0178] Three kinds of composite antioxidants were added to milk powder samples in different proportions, and the mean value of the total content of oxidized flavor substances (hexanal, nonanal, and 2-heptanone) was used as an evaluation index. The total content of oxidized flavor substances detected by the instrument is shown in Table 3. From the results in Table 3, it can be seen that the total content of oxidized flavor substances is greater than 29.00 μg / kg in: Examples 1, 2, 3, 5, and 9; the total content of oxidized flavor substances is between 28.00 μg / kg and 29.00 μg / kg in: Examples 4, 7, 8, and 12; the total content of oxidized flavor substances is between 27.00 μg / kg and 28.00 μg / kg in: Example 13; the total content of oxidized flavor substances is less than 27.00 μg / kg in: Examples 6, 10, 11, 14, 15, and 16.

[0179] In summary, from the comparison between the embodiments and the comparative examples, it can be concluded that by rationally compounding the three natural antioxidants, a synergistic effect of improving the stability of oxidized flavor substances in milk powder can be obtained compared with the use of only one antioxidant and the compounding of two antioxidants, and the rational use of these three antioxidants according to the ratio of the present invention can more accurately determine and more reasonably reflect the content of oxidized flavor substances in milk powder during the shelf life.

[0180] Industrial Applicability

[0181] The natural antioxidant of the present invention can be used industrially.

Claims

1. An application of a compound antioxidant in the detection of oxidative flavor substances in milk powder products, characterized in that: The compound antioxidant contains L-ascorbic acid, ascorbyl palmitate and α-tocopherol as active ingredients. The mass ratio of the three active ingredients in the composite antioxidant is: L-ascorbic acid: ascorbyl palmitate: α-tocopherol = (2-3.5): (2-7): 1; Based on the total mass of the milk powder product, the contents of L-ascorbic acid, ascorbyl palmitate and α-tocopherol in the composite antioxidant are 0.0036% to 0.03% by mass, 0.0036% to 0.061% by mass, and 0.0036% to 0.0087% by mass, respectively. Based on the total mass of the milk powder product, the added amount of the compound antioxidant is 0.01 mass % to 0.1 mass %.

2. The use according to claim 1, characterized in that: The mass ratio of L-ascorbic acid, ascorbyl palmitate and α-tocopherol in the composite antioxidant is: L-ascorbic acid:ascorbyl palmitate:α-tocopherol=(2.5-3.5):(3-4):

1.

3. A method for testing oxidized flavor substances in milk powder products, characterized in that: The test method includes: Pre-processing steps, The extraction steps, The steps of detection, Wherein, in the pre-treatment step, a compound antioxidant is added to the milk powder product to be tested to obtain a test sample; The compound antioxidant contains L-ascorbic acid, ascorbyl palmitate and α-tocopherol as active ingredients. The mass ratio of the three active ingredients in the composite antioxidant is: L-ascorbic acid: ascorbyl palmitate: α-tocopherol = (2-3.5): (2-7): 1; Based on the total mass of the milk powder product, the contents of L-ascorbic acid, ascorbyl palmitate and α-tocopherol in the composite antioxidant are 0.0036% to 0.03% by mass, 0.0036% to 0.061% by mass, and 0.0036% to 0.0087% by mass, respectively. Based on the total mass of the milk powder product, the added amount of the compound antioxidant is 0.01 mass % to 0.1 mass %; The extraction step includes extracting the test sample by a solid phase extraction method to collect flavor substances.

4. The testing method according to claim 3, characterized in that: The detection step includes detecting the flavor substances collected in the extraction step using chromatography and mass spectrometry.

5. Application of the test method for oxidized flavor substances in milk powder products according to claim 3 or 4 in the field of sensory evaluation of milk powder.

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