Review Study on Determination of Food Additives in Dairy Products

Food additives are materials incorporated into food items to improve their quality according to their standards. In the dairy sector, additives are significant in ensuring product stability and consumer acceptability. Because dairy products like milk, cheese, yogurt, and cream are perishable, additives inhibit spoilage, ensure uniform texture, and maintain nutritional content throughout storage and transit. [2] Some common food additives present in dairy products are preservatives (such as potassium sorbate), stabilizers (such as carrageenan, guar gum), emulsifiers, colouring agents, flavour enhancers, and sweeteners. These maintain the required viscosity in items such as flavoured milk or yogurt, inhibit syneresis (separation of water) in milk gels, and ensure homogeneous texture in ice cream or cheese spreads. Fortified additives such as vitamins A and D are also added to enhance nutritional value. [2] All food additives utilized in dairy need to adhere to food safety laws set by agencies like the FDA, EFSA, or FSSAI. Although their application is typically harmless within set limits, increasing consumer demand for clean-label offerings has manufacturers looking towards natural substitutes. [3]

Dairy Products:

Dairy foods are commodities that come from the milk of mammals, the bulk of which comes from cows, but also goats, sheep, and buffalo. These foods are all highly nutritious and contain such essential nutrients as calcium, protein, vitamin D, and potassium, which make them vital to maintaining bone structure, muscles, and general growth and development. [4] There are quite a number of different dairy products, each produced by a particular process such as fermentation, churning, or aging. The most widely known ones are:

1. Milk– This is the simplest form of dairy product and is consumed in numerous forms: whole, skimmed, semi-skimmed, or flavoured. Milk serves as a foundation used to produce other dairy products. [5]

2. Cheese – Cheese is prepared by curdling milk and dividing the solid curds from the liquid whey. There are hundreds of types, like cheddar, mozzarella, gouda, and brie, that vary in texture, flavour, and aging process. [6]

3. Yogurt – Yogurt is prepared by fermenting milk with special bacterial cultures. It is of creamy consistency and may be eaten plain or sweetened. Greek yogurt, which is strained to eliminate whey, is thicker and more protein-rich.

4. Butter – Butter is produced by churning cream to isolate the fat from the buttermilk. It is employed in cooking, baking, and as a spread. Its types include salted, unsalted, and cultured butter.

5. Cream – Cream is the skimmed high-fat layer of milk. It exists in various forms such as heavy cream, sour cream, and whipped cream, and is utilized in desserts, sauces, and soups.

6. Ice Cream – A frozen dairy dessert prepared from cream, sugar, and flavourings, usually churned to form a smooth texture. [5,6,7]

Food additives:

Food additives are added to food to maintain flavour, taste, appearance, or shelf life. They encompass preservatives, colorants, flavourings, and emulsifiers. In minute amounts, they contribute to the safety, quality, and attractiveness of food. All the additives have to be approved and governed to guarantee the health and safety of consumers. [2]

Classification:

1.Classification Based on Function:

1) Preservatives: Preservatives prevent microorganism spoilage of dairy products (e.g., sodium benzoate, nitrates). [2,8]

2) Antioxidants: Antioxidants prevent rancidity and oxidation (e.g., BHA, BHT, ascorbic acid).

3) Colorants:  Colorants add or restore colour (e.g., tartrazine, caramel colour).

4) Flavouring Agents: Flavouring agents add or intensify flavour (e.g., vanillin, monosodium glutamate).

5) Sweeteners: Sweeteners substitute sugar (e.g., aspartame, saccharin, stevia).

6) Emulsifiers: Emulsifiers mix oil and water (e.g., lecithin, mono- and diglycerides).

7) Stabilizers: Stabilizers help with texture and consistency (e.g., carrageenan, xanthan gum).

8) Thickeners: Thickeners are used to thicken (e.g., pectin, starch, guar gum).

9) Anti-caking Agents: Anti-caking Agents are used to prevent clumping (e.g., calcium silicate, magnesium carbonate).

10) Acidulants: Acidulants add or control acidity (e.g., citric acid, acetic acid).

11) Leavening Agents: Leavening Agents release gases to increase volume (e.g., baking powder, sodium bicarbonate).

12) Humectants: Humectants retain moisture (e.g., glycerol, sorbitol).

13) Sequestrants: Sequestrants bind metal ions to improve quality (e.g., EDTA, citric acid).

14) Glazing Agents: Glazing Agents give a shiny appearance or coating (e.g., beeswax, shellac).

15) Nutritional Additives: Nutritional Additives add nutrients (e.g., vitamins, minerals). [2,8]

2. Classification Based on Origin:

Types and Examples

1. Natural: Naturally derived from natural sources.

Ex: Beet juice (colour), lecithin. [2,9,10]

2) Synthetic (Artificial): Man-made but not naturally occurring. Ex: Saccharin, tartrazine.

Nature-Identical Chemically produced but indistinguishable from natural ones. Vanillin (synthetic), ascorbic acid. [2,9,10]

3. Classification Based on Chemical Nature:

Types and Examples:

Organic compounds: Sorbic acid, citric acid, benzoic acid. [11]

Inorganic compounds: Sodium nitrate, phosphates, calcium salts. [11]

Enzymes: Amylase, protease (used in processing). [10]

Gums and polysaccharides: Guar gum, xanthan gum, pectin. [12,13]

Harmful Effects of food additives in dairy products:                                                                                                                                                                                                              

Food additives are used in dairy products to improve flavour, texture, shelf life, and appearance. But some of these food additives have adverse effects on health, particularly with prolonged intake or on sensitive subjects.  [2]                          

Below are adverse effects of popular food additives in dairy products:

General Analytical Methods for Determination of Food Additives in Dairy Products:

1. Sample Preparation (First Critical Step)

Prior to analysis, any dairy product must be prepared due to its fat, protein, and water composition. [20]

Common techniques:

Extraction:

 Extraction involves the separation of the analyte from the sample matrix by its transfer into a suitable solvent or phase according to its different solubility. [20]

Two types:

1.Liquid-Liquid Extraction,

2.Solid-Liquid Extraction.

Liquid–Liquid Extraction (LLE): The separation of analytes between two immiscible liquids. [23]

Solid-Liquid Extraction (SLE): The process of extraction of analytes from solid matrices by using any liquid solvent. [25]

Protein Precipitation (By acids/solvents)

Protein precipitation usually refers to the process of removing proteins from a sample by addition of acids, salts, or organic solvents in order to avoid an analytical interference. [22]

Defatting: (e.g., with hexane)

Defatting refers to the removal of fats in dairy samples using non-polar solvents, such as hexane, to provide better accuracy in analysis. [23]

Filtration or centrifugation:

Filtration is defined as a physical method of separating solid particles from a liquid by passing the liquid mixture through a porous medium [22].

Derivatization (making some additives traceable):

Derivatization is based on the chemical modification of an analyte to enhance its detectability or volatility.

2. Chromatographic Methods (Most Widely Used)

a) High-Performance Liquid Chromatography:

HPLC is a separation technique where the chemical identity of the desired component or compound is determined, separated, and quantified on the basis of its interaction with the packing material, which passes through a column at high-pressure liquid flow. HPLC is based on the differential interaction between sample components and the two phases involved in the process, which are: a mobile phase (liquid solvent), and a stationary phase (solid particles inside the column). Those compounds that have a greater affinity for the stationary phase will migrate slower, and this leads to the separation of the compounds. It is a liquid phase separation method that employs high pressure. It separates non-volatile and thermally labile compounds. [26,27]

Used for:

Preservatives: These are added to inhibit microbial activity.

Ex: sorbates, benzoates. [26]

Sweeteners: Additives used as sweetening agents.

Ex: Aspartame. [26]

Colorants: Colorants are substances that are added to a material to restore colour.

Ex: Tartrazine, Sunset Yellow, and Indigo Carmine. [26]

Antioxidants: Agents that counteract oxidation.

Ex: Phenolic acids—gallic, caffeic, and rosmarinic acid; flavonoids—quercetin, rutin, and EGCG; vitamins C and E; and synthetic antioxidants—BHA and BHT. [26]

Advantages:

These are High accurate and sensitive and Applicable to non-volatile substances

b) Gas Chromatography (GC):

GC is a method used for the separation of volatile substances with a gaseous mobile phase. Gas Chromatography (GC) is an analytical separation technique applied for the separation and quantification of volatile and semi-volatile compounds by transporting the compounds as a gas through a stationary phase in a column. GC separates substances based on the following principles of a compound according to GC: Volatility, and Interaction with the stationary phase. More volatile molecules will elute first, and less volatile molecules will take longer to elute through the column. [28]

Used for:

Volatile additives,

Flavouring agents,

Some Preservatives (After Derivatization) [29]

Advantages:

High separation efficiency [30]

3. Spectroscopic Methods

a) UV-Visible Spectrophotometry:

Ultraviolet and visible spectrophotometry is the analytical technique that determines the identity and amount of sample by the amount of ultraviolet or visible radiation it absorbs by a solution. This technique is based on the "Beer-Lambert law," which suggests that the absorbance of a substance is proportional to concentration and the path of the light. [31] Applied for Artificial colorants, Nitrites/nitrates and for some preservatives.

Benefits:

This is Easy and inexpensive method and used for Rapid analysis. [31]

Limitations:

Not as selective as chromatography. [31]

b) Infrared Spectroscopy (FTIR) or Fourier Transform Infrared Spectroscopy:

Fourier Transform Infrared Spectroscopy, or FTIR, is a kind of analytical method used to help identify chemicals based on their absorption characteristics upon infrared radiation. This radiation is responsible for the vibration of molecules. FTIR is based on the fact that different chemical bonds will absorb certain infrared wavelengths.” The spectrum produced is a molecular fingerprint of the compound. Used for Screening and authentication and for Adulteration detection/additive presence detection.

Benefits:

 It requires small sample and Non-Destructive. [32]

4. Mass Spectrometry (MS)

Mass Spectrometry is a method of analysis used to determine the identity of a sample based on the mass-to-charge ratio of ions. In mass spectrometry, a substance is changed to gas ions. The gas ions produced are then separated based on the mass-to-charge ratio (m/z) after being accelerated. The separated gas ions are then detected, forming a mass spectrum that aids in the identification of the substance and the determination of the molecular weight of the substance.

Often coupled with GC or HPLC:

GC-MS

LC-MS/MS,

Used for:

Trace-level detection,

Confirmation of additive identity

Advantages:

High sensitivity, high specificity. [33]

5. Electrochemical Methods:

Electrochemical methods are those techniques of analysis that carry out the measurement of electrical signals (current, voltage, or charge) produced from a chemical reaction happening at the electrode surface. These are related to the concentration of analytes in samples directly.

Used for Preservatives, Antioxidants, Sweeteners.

Advantages:

This method is Fast and cost-effective and Applicable for routine analysis. [34]

6. Immunochemical Methods:

Immunochemical techniques involve analytical procedures that utilize specific antigen-antibody reactions, making it possible to identify food additives as well as contaminants in dairy products. Immunochemical techniques have the highest sensitivity and selectivity, allowing the detection of food additives even in trace amounts. Immunochemical techniques include ELISA (Enzyme-Linked Immunosorbent Assay) as well as rapid test kits. The primary aim of immunochemical techniques is detection and verification of additives which promotes the safety of foods. [35]

Used for Specific additives or contaminants.

Advantages:

This method is High selectivity and Applicable to large number of samples only.

Limitations:

Requires specific antibodies. [35]

7. Capillary Electrophoresis:

Capillary electrophoresis is an analytical procedure employed to separate food additives found in dairy products based on the use of the charge and size of the molecules that are affected by an electric field within the walls of the narrow-bored capillary tube. On the application of the electric voltage, the molecules move at different rates based on their electrophoretic mobilities.  Basically, capillary electrophoresis is employed to separate additives such as preservatives, organic acids, sweeteners, and colorants. Primarily, the objective of performing capillary electrophoresis is the separation or quantification of food additives at low volumes using the electric voltage. Used for determination of Organic acids, Preservatives, Sweeteners. The aim of Capillary Electrophoresis is the rapid separation, identification, and quantification of food additives in dairy products according to charge and size, using high electric fields, with high efficiency, high accuracy, and low sample volumes.

Benefits:

For Quick Separation and Low solvent consumption. [36]

LITERATURE REVIEW: