1. The Enzymatic Method and the Importance of Determining Acid Content in Food
1.1. The Enzymatic Method
- Enzymatic tests are essential analytical tools used to determine sugars, acids, alcohol and other food components in many product categories, including fruit juice, wine, beer, dairy products, eggs and meat.
- These tests are highly specific and effective even in complex sample matrices, thanks to high-quality enzymes that ensure accurate and precise measurements. The results of these enzymatic tests are measured with a spectrophotometer and the process can be automated for maximum efficiency.
1.2. The Importance of Determining Acid Content in Food
- Some acids can spoil products, some others are used as additives and some others still serve as important quality indicators.
- Determining acid content in food helps ensure the stability of flavour and the nutritional value of the food. Determining the level of organic acids such as citric acid, lactic acid and acetic acid is very important in beverage production such as wine and beer. This work ensures that foods and beverages made for consumers have uniform, well-defined characteristics, including shelf life.
- Understanding the role of these acids in food production is necessary to maintain product quality and safety.
1.3. Certification and Approval by International Organizations
Many enzymatic methods have been validated or approved by leading international organizations, ensuring their reliability and standardization in food analysis. The most important of these organizations include:
- AOAC (Association of Official Analytical Chemists)
- CEN (European Committee for Standardization)
- IDF (International Dairy Federation)
- IFU (International Federation of Fruit Juice Producers)
- ISO (International Organization for Standardization)
- OIV (International Organisation of Vine and Wine)
2. Significance of the Acids Present in Food
2.1. Acetic Acid
- Acetic acid is an important analytical metabolite, the end product of fermentation and the oxidation product of acetaldehyde and ethanol. Determination of acetic acid is particularly important in the wine industry: as a sensory compound, it enhances flavour and complexity in small amounts, but spoils the wine at high concentrations.
- Acetic acid is the main component of "volatile acidity" in wine and is measured throughout the wine fermentation process. Vinegar contains about 3 - 9% acetic acid by volume and gives this product economic value. Acetic acid is also used in food production as a preservative (E260) and as a flavour improver
- Therefore, it is tested in many food product types such as beer, bread and bakery products, leavening agents and sourdough, milk, fish, fruit and vegetable products, meat, ketchup, mayonnaise, mixed pickles, salt and spices, sauces and dressings, soy sauce, tea and vinegar, as well as in animal feed and pharmaceuticals.
* The Acetic Acid rapid test kits from R-Biopharm include the following products:
- Enzytec™ Liquid Acetic acid (Art. No. E8226)
- RIDA®CUBE Acetic acid (Art. No. RCS4226)
- Enzytec™ Generic Acetic acid (Art. No. E1226)
- Enzytec™ Multi-acid standard low & high (Art. No. E8460 / E8465)
2.2. L-Ascorbic Acid (Vitamin C)
- L-ascorbic acid (vitamin C) is a natural organic compound with antioxidant properties that is present in fruits and vegetables. Quantitative determination is particularly important in the production of wine, beer, milk, beverages and fruit juice. With its essential role in the human diet, L-ascorbic acid (E300) and its salt derivatives (E301-303) are often used as food additives, with the added benefit of antioxidant properties and flavour enhancement.
- In the wine industry, L-ascorbic acid can be added to prevent the oxidation of the wine. In brewing, L-ascorbic acid and its salts are effective antioxidants and have a positive effect on flavour, taste and stability
- In the meat industry, L-ascorbic acid is used as a colour fixative and antioxidant. L-ascorbic acid is also used in the pharmaceutical industry and as an additive in animal feed. If L-ascorbic acid is added in food production, its content must be controlled.
* L-Ascorbic Acid (Vitamin C) rapid test kits from R-Biopharm:
Enzytec™ Generic L-Ascorbic acid (Art. No. E1267)
2.3. Citric Acid
- Citric acid is a very important metabolite in animals, plants and microorganisms. It is the best known of the fruit acids and occurs in high amounts, for example in citrus fruits (about 10 g citric acid/litre of orange juice). Citric acid is produced biotechnologically on a large scale as a food additive (E330).
- Citric acid is used as a preservative (acidulant) for food or cosmetics, as a metal chelating agent (for example iron in wine), as an emulsifier (for example in processed cheese production) and as a flavouring agent in beverage and confectionery production. It is also a component of many pharmaceuticals and detergents.
* The Citric Acid rapid test kits from R-Biopharm include the following products:
- Enzytec™ Liquid Citric acid (Art. No. E8230)
- Enzytec™ Generic Citric acid (Art. No. E1214)
- Enzytec™ Multi-acid standard low & high (Art. No. E8460 / E8465)
2.4. Formic Acid
- Formic acid occurs as a metabolite in many biochemical reactions, but its concentration is always very low. It is the oxidation product of methanol and formaldehyde. Because formic acid at low concentrations has both bactericidal and fungicidal effects, it can be used as a food preservative. Molds tend to produce formic acid as a metabolite; therefore, determination of formic acid can give an indication of properties such as the degree of decomposition of samples
- Formic acid belongs to the hazardous substances in yeast production. Formic acid is a by-product in the acetic acid fermentation process in vinegar production and is also part of the "volatile acidity" in wine.
* Formic Acid rapid test kit from R-Biopharm:
Enzytec™ Liquid Formic acid (Art. No. E8210)
2.5. Gluconic Acid
- Gluconic acid occurs naturally: in fruits, honey and wine, but also in meat or dairy products. As a food additive (E574), it is an acidity regulator. It is also used in cleaning products, where it dissolves deposited minerals. This acid gives a sour but refreshing taste. For the wine industry, grapes infected with Botrytis show an increase in this acid that can reach 1-2 g/L
- D-Glucono-δ-lactone is found together with D-gluconic acid, for example in wine, and is also widely used in the food industry (yoghurt, cheese, bread, confectionery, meat and sausages) to control acidity.
* The Gluconic Acid rapid test kits from R-Biopharm include the following products:
- Enzytec™ Liquid D-Gluconic acid (Art. No. E8520)
- Enzytec™ Generic D-Gluconic acid (Art. No. E1223)
- Enzytec™ Multi-acid standard low & high (Art. No. E8460 / E8465)
2.6. Glutamic Acid
- L-glutamic acid (L-glutamate), one of the 20 common amino acids, occurs naturally in foods such as cheese, milk, meat, fish, maize, tomato, mushrooms, soybeans and sugar beet. Knowledge of the L-glutamate content is of great interest, for example in the ripening process of plants, in the liver used to produce food for humans and in controlling the ripening of cheese.
- L-glutamic acid is one of the main components that enhance the flavour of food (Monosodium glutamate; MSG). Glutamate is an allergen; excessive use of MSG as a food additive (E621) can lead to the symptoms of “Chinese restaurant syndrome”.
* Glutamic Acid rapid test kit from R-Biopharm:
Enzytec™ Liquid L-Glutamic acid (Art No. E8530)
2.7. D-3-Hydroxybutyric Acid
- The increase in the content of D-3-hydroxybutyric acid in eggs can be detected 6 days after the hens are inseminated. This increase continues even after the embryo has died
- Therefore, D-3-hydroxybutyric acid is a typical indicator for fertilized and incubated eggs up to 10 mg/kg (legal limit) and can reach 800 mg/kg in heavily spoiled eggs. In egg and bakery products, the hydroxybutyric acid level is an indicator of how fresh the eggs are when they are used.
* D-3-Hydroxybutyric Acid rapid test kit from R-Biopharm
Enzytec™ Generic D-3-Hydroxybutyric acid (Art. No. E2610)
2.8. D-Isocitric Acid
- D-isocitric acid is part of the citric acid cycle and is therefore present in all animal and plant species. Its content is usually very low. Determination of D-isocitric acid has become important in fruit juice analysis (especially orange juice) to detect illegal additives (for example citric acid). Not only is the minimum D-isocitric acid content in juice of interest, but the ratio of citric acid to D-isocitric acid is also very important: A ratio that is too high indicates addition of citric acid
- For example, in pure orange juice, the ratio of citric acid to D-isocitric acid is usually less than 130 and higher values may indicate adulteration of the fruit juice. The D-isocitric acid content in products containing orange juice can also be used to calculate the orange juice content, for example in beverages labelled on the bottle as "contains orange juice".
* The D-Isocitric Acid rapid test kits from R-Biopharm include the following products:
- Enzytec™ Liquid D-Isocitric acid (Art. No. E8550)
- Enzytec™ Generic D-Isocitric acid (Art. No. E1222)
2.9. D-/L-Lactic Acid
- D-lactate is usually measured together with L-lactate and is almost never measured on its own. D-lactic acid is only formed by certain microorganisms, for example by Lactobacillus lactis , Lb. bulgaricus and Leuconostoc cremoris
- D-lactic acid is not formed, or is only formed in trace amounts, by "higher organisms", for example by animals. Therefore, the presence of D-lactate can serve as an indicator of contamination or spoilage, assuming that fermentation techniques have not been used in food production.
* The D-/L-Lactic Acid rapid test kits from R-Biopharm include the following products:
- Enzytec™ Liquid D-/L-Lactic acid (Art. No. E8240)
- RIDA®CUBE D-/L-Lactic acid (Art. No. RCS4240)
- Enzytec™ Generic D-/L-Lactic acid (Art. No. E1255)
- Enzytec™ Multi-acid standard low & high (Art. No. E8460 / E8465)
2.10. D-Lactic Acid
- D-lactate is very often measured together with L-lactate and is almost never measured on its own. D-lactic acid is only formed by certain microorganisms, for example by Lactobacillus lactis, Lb. bulgaricus and Leuconostoc cremoris. D-lactic acid is not formed, or is only formed in trace amounts, by "higher organisms", for example by animals
- Therefore, the presence of D-lactate can serve as an indicator of contamination or spoilage, assuming that fermentation techniques have not been used in food production, for example in the production of yoghurt products.
* The D-Lactic Acid rapid test kits from R-Biopharm include the following products:
- Enzytec™ Liquid D-Lactic acid (Art. No. E8245)
- Enzytec™ Multi-acid standard low & high (Art. No. E8460 / E8465)
2.11. L-Lactic Acid
- L-lactic acid is present in many foods and beverages. Produced naturally by lactic acid bacteria, L-lactic acid is present in many fermented dairy products such as yoghurt, and also in pickles, cured meat and fish. It is often added to foods and beverages (E270) as a non-volatile acidulant. In the wine industry, the malolactic fermentation process is monitored by following the decrease in L-malic acid level and the increase in L-lactic acid level. The L-lactate content in beer indicates the presence of Lactobacilli in production. The L-lactate content in liquid whole egg or in egg powder provides good information on the hygiene status of the product.
- Similarly, the quality of milk and of fruit and vegetables can be established by measuring the L-lactic acid content. L-lactate in milk powder indicates the use of neutralized yoghurt in milk powder production. L-lactate in wine is also formed during "secondary fermentation" (malo-lactic fermentation).
* The L-Lactic Acid rapid test kits from R-Biopharm include the following products:
- Enzytec™ Liquid L-Lactic acid (Art. No. E8260)
- RIDA®CUBE L-Lactic acid (Art. No. RCS4260)
- Enzytec™ Generic L-Lactic acid (Art. No. E1254)
- Enzytec™ Multi-acid standard low & high (Art. No. E8460 / E8465)
2.11. D-Malic Acid
- D-malic acid in fact does not occur in nature; it is a metabolite that is only produced by certain microorganisms. Analysis of fresh fruit juice shows the presence of D-malic acid at the detection limit. Therefore, the legal limit for fruit and fruit juice is recommended to be 10 mg/l. D-malic acid is a component of D-/L-malic acid (racemic) when it is processed chemically.
- Since natural products in fact do not contain D-malic acid, the detection of D-malic acid indicates that D-/L-malic acid has been added, for example to wine or fruit juice, which may be permitted or prohibited depending on the country.
* D-Malic Acid rapid test kit from R-Biopharm:
Enzytec™ Liquid D-Malic acid (Art No. E8270)
2.12. L-Malic Acid
- L-malic acid is one of the most important fruit acids and the quantitative determination of L-malic acid is particularly important in the production of wine, beer, bread, fruit and vegetable products, as well as in cosmetics and pharmaceuticals. The bacterial breakdown of L-malic acid leads to the formation of L-lactic acid, which may be the desired reaction in wine production (malo-lactic fermentation, biological acid reduction) or the undesired reaction in the case of beer (secondary fermentation).
- L-malic acid also has many applications as a food preservative (E296) because it is a stronger acid than citric acid and is a flavour-enhancing compound.
* The L-Malic Acid rapid test kits from R-Biopharm include the following products:
- Enzytec™ Liquid L-Malic acid (Art. No. E8280)
- RIDA®CUBE L-Malic acid (Art. No. RCS4280)
- Enzytec™ Generic L-Malic acid (Art. No. E1215)
- Enzytec™ Multi-acid standard low & high (Art. No. E8460 / E8465)
2.13. Oxalic Acid
- Oxalic acid can occur at high concentrations in leaves. Oxalate in food affects the oxalate level in urine and in diseases its presence can lead to the formation of calcium oxalate stones in the bladder and kidneys. Moreover, oxalate in food inhibits reabsorption and therefore the uptake of calcium, and is therefore of considerable nutritional importance.
- Spinach, peanuts, chocolate, rhubarb and tea contain high oxalate contents. Foods with a high oxalate content should not be included in the diet of growing children (spinach!), pregnant women, people with a calcium deficiency and patients who have passed calcium oxalate stones.
- Oxalic acid in beer, especially calcium oxalate, is the cause of foaming, hazing and precipitation.
* Oxalic Acid rapid test kit from R-Biopharm:
Enzytec™ Generic Oxalic acid (Art. No. E2100)
2.14. Succinic Acid
- Succinic acid is a metabolite of the citric acid cycle, succinic acid is abundant in animals, plants and microorganisms. Succinic acid is a specific indicator of bacterial breakdown in eggs and egg products (> 5 mg/kg).
- Succinic acid concentration is also monitored during the production of many food and beverage types, including wine, soy sauce, soy flour, fruit juice and dairy products (for example cheese), and is also used as a flavouring agent. The ripening process of apples can be monitored by following the gradual decrease in succinic acid level.
- Succinic acid is also used in many non-food applications (dyestuffs, pharmaceuticals, perfumes, lacquers, coolants).
* Succinic Acid rapid test kit from R-Biopharm:
Succinic acid (Art. No. 10176281035)
2.15. Tartaric Acid
- Tartaric acid is one of the main acids in grapes and is therefore an important parameter in wine production.
* Tartaric Acid rapid test kit from R-Biopharm:
Enzytec™ Color Tartaric acid (Art. No. E3100)
3. Main benefits of monitoring acid content in food components
- Precise component analysis: Ensures accurate measurement of sugars, acids, alcohol and other food components, maintaining product quality and consistency.
- Standards compliance: Helps manufacturers meet international standards and regulations, such as those set by AOAC, CEN, IDF, IFU, ISO and OIV.
- Quality control: Strengthens quality control processes, ensuring products meet the desired specifications and consumer expectations.
- Versatility: Suitable for many food types, including fruit juice, wine, beer, dairy products, eggs and meat, making this a useful tool for many segments of the food industry.
- Easy to automate: Creates favourable conditions for automation in the laboratory, improving efficiency and accuracy in routine analysis.
Monitoring food components is necessary to maintain the quality, safety and compliance of food products. Using advanced enzymatic tests and technologies such as the SureFood® and Enzytec™ product lines, manufacturers can achieve accurate and reliable measurement of key food components. This not only ensures product consistency and consumer satisfaction but also helps meet strict international standards and regulations. Implementing comprehensive component testing supports strong quality control and improves overall production efficiency.