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Course:FNH200/Projects/2025/Apple Cider Vinegar

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Introduction

Long a by-product of winemaking, vinegar began to be produced commercially at scale in Orléans in the 17th century, helping cement its role in European cookery.

Apple cider vinegar (ACV), also known as cider vinegar, is a vinegar made from fermented apple juice. It is commonly used in salad dressings, marinades, vinaigrettes, food preservation, and various condiments such as chutneys [1]. The production of ACV involves a natural, two-step fermentation process using specific microorganisms that help convert sugars into vinegar.

Vinegar is among the oldest known fermented products. Wine likely came first; vinegar was produced when wine soured and cider is the alcoholic drink made by fermenting apple juice. Cider vinegar (apple cider vinegar) is the vinegar obtained when alcoholic cider is converted to acetic acid by acetic acid bacteria [2].

Alcoholic beverages and vinegars have been used since antiquity across many cultures for drinking, preservation, and medicine. Earlier civilizations weren’t aware of the microbiology, but they recognized the effects of alcohol and souring. Written records note wine production in Mesopotamia and Egypt by about 5000 BCE. The term vinegar comes from French vinaigre which roughly means “sour wine” [2].

Fermentation Process of Apple Cider Vinegar

Step 1: Alcoholic Fermentation

The first stage of fermentation is known as alcoholic fermentation. In this anaerobic (without oxygen) process, natural or added yeasts, mainly Saccharomyces cerevisiae, convert the sugars in crushed apple juice into ethanol (alcohol) and carbon dioxide (CO₂). This stage transforms sweet apple juice into an alcoholic beverage, commonly known as hard cider [3][4].

A diagram representing the Apple Cider Vinegar Fermentation Process.

C₆H₁₂O₆ = Glucose (sugar) | C₂H₅OH = Ethanol (alcohol) | CO₂ = Carbon dioxide (gas)

Step 2: Acetic Acid Fermentation

The second stage is acetic acid fermentation. In this aerobic (oxygen-present) phase, Acetobacter bacteria, especially Acetobacter aceti, convert the ethanol produced in the first stage into acetic acid. This gives vinegar its sour flavour and acidic properties. These bacteria thrive in acidic environments and use oxygen to complete the conversion process [3][5].

C₂H₅OH = Ethanol | O₂ = Oxygen | CH₃COOH = Acetic acid (main component of vinegar) | H₂O = Water

Micro-organisms involved in the Fermentation Process

Yeasts (Alcoholic Phase)

Acetic Acid Bacteria (AAB) used in the production of acetic acid from alcohol and oxygen.

During alcoholic fermentation, yeasts are the primary microorganisms responsible for turning sugars into alcohol. The most common yeast used is Saccharomyces cerevisiae, although other strains like Lactococcus may also be involved. These yeasts can be natural (wild) or commercial strains, and they also help develop flavor compounds during the fermentation process [3][6][7].

Acetobacter (Acetic Phase)

In the second stage of fermentation, bacteria from the Acetobacter genus play a major role. The most studied species is Acetobacter aceti. These bacteria are aerobic, meaning they require oxygen to function. They convert ethanol to acetaldehyde and then to acetic acid using specific enzymes. These bacteria are also acid-tolerant, which allows them to survive in the acidic environment of vinegar, with acidity levels reaching 5–20% [6][7][8].

Packaging and Shelf Life of Apple Cider Vinegar

Apple cider vinegar is naturally shelf-stable due to its high acetic acid content, which inhibits microbial growth and slows enzymatic activity. However, packaging plays an important role in maintaining the product quality over time i.e. protecting the vinegar from light, oxygen, moisture, and physical contamination, all of which can contribute to chemical changes and sensory deterioration.

Impact of Packaging on Product Quality

A bottle of apple cider vinegar next to apples for display.

Glass bottles are the most common packaging which are chemically inert, non-porous, and impermeable to gases and vapours. Glass protects against oxygen ingress and is critical for minimizing oxidative reactions such as flavour degradation or the loss of volatile aroma compounds. Additionally, glass acts as an effective barrier to moisture transfer and does not leach substances into the product, unlike some plastics, which can interact with acidic liquids over time [9]. Sealing integrity is equally important. Tight-fitting caps with liners or heat-induction seals prevent oxygen exposure, which otherwise accelerates oxidative changes. Exposure to oxygen can also lead to the formation of additional cloudiness from microbial “mother” growth in raw, unpasteurized vinegars, even if the product remains safe to consume [10]. Packaging must also provide light protection. According to principles of oxidative deterioration, light can accelerate vitamin degradation (particularly vitamin C) and promote oxidative rancidity in products containing even trace amounts of residual oils from apples. Amber or tinted glass is therefore preferred for premium ACV to reduce photooxidation [11]. Any sustainable packaging alternative must still provide adequate barrier properties to oxygen and moisture, maintain structural integrity during handling, and avoid undesirable interactions with the product.

Shelf Life Considerations

Due to its acidity (pH typically ~3), unopened ACV has an almost indefinite shelf life, as microbial growth is effectively inhibited. Commercial guidelines suggest optimal quality retention for 2–5 years unopened and 1–2 years after opening if stored correctly . While spoilage is rare, quality changes, such as sediment formation, flavor softening, and slight acidity loss, can occur with prolonged oxygen exposure or temperature fluctuations [12].

Best practices for storage include:

  • Keeping bottles tightly sealed when not in use
  • Storing in a cool, dark location away from sunlight and heat sources
  • Avoiding repeated temperature cycling, which can accelerate chemical changes

Health Claims and Scientific Evidence

Apple cider vinegar has been praised for its potential to support gut health [13], help regulate blood sugar [14], and provide antimicrobial benefits [15]. Raw, unfiltered ACV contains acetic acid and natural probiotics [16], which can encourage the growth of beneficial gut bacteria and promote better digestion. Its acidity may also slow the rate at which food leaves the stomach, allowing for better nutrient absorption and smoother digestive functions [13]. ACV has been shown to help moderate blood sugar levels, particularly after meals, by slowing carbohydrate absorption and improving insulin sensitivity, making it a helpful addition for supporting stable blood sugar throughout the day [14]. Another notable benefit is its antimicrobial properties, as ACV can inhibit the growth of harmful bacteria, yeasts, and molds, which has contributed to its long history of use in food preservation and natural cleaning. These effects may also support oral and digestive health [15]. When used in moderation, ACV can be a simple way to enhance overall wellness. However, it should be diluted to prevent tooth enamel erosion and digestive irritation, with a typical safe intake being 1 to 2 tablespoons in water daily. This makes ACV a versatile addition to a balanced lifestyle offering both traditional and research-supported health advantages [17].

Food and Safety Regulations of Apple Cider Vinegar

In Canada, vinegar,  including apple cider vinegar (ACV) , is a standardized food regulated under the Food and Drug Regulations (FDR) within the Food and Drugs Act. Standards of identity and composition specify the ingredients and characteristics a product must have to be legally sold as “vinegar,” such as a minimum acetic acid concentration [18]. The Health Products and Food Branch (HPFB) of Health Canada sets these standards, while the Canadian Food Inspection Agency (CFIA) enforces them through inspections, labelling oversight, and compliance measures [19].

Under the FDR, vinegar must be safe, unadulterated, and manufactured under sanitary conditions (Sections 4 and 7 of the Act) [20]. Pre-packaged vinegar must meet labelling requirements under the Consumer Packaging and Labelling Act, including bilingual common name, net quantity, manufacturer information, and ingredient listing [21]. If health claims are made (e.g., “supports digestion”), they must meet strict evidence-based criteria and cannot imply treatment or prevention of diseases listed in Schedule A, such as diabetes or arthritis [22].

Pasteurized vs. Raw / Unfiltered ACV

Mother of Vinegar; a biofilm of bacteria, cellulose and yeast.

Pasteurized ACV has been heat-treated to destroy potentially harmful microorganisms, increasing shelf life and ensuring safety for the general population. Raw or unfiltered ACV contains the “mother” – a complex of acetic acid bacteria and cellulose – and is often marketed as a natural product. While raw ACV can be safe, it may carry a higher microbial load. Canadian regulations require that all vinegar, whether pasteurized or raw, meet microbiological safety standards under the Food and Drugs Act, including the absence of pathogens such as Escherichia coli or Salmonella [18][22].

International Comparisons

Internationally, standards vary. The Codex Alimentarius Commission, of which Canada is a member, sets voluntary guidelines for vinegar identity and safety to promote fair trade and consumer protection [23]. In the United States, the Food and Drug Administration (FDA) regulates vinegar under federal food law, with similar acetic acid concentration requirements but broader definitions of what constitutes a food additive [24]. Some countries mandate pasteurization for all commercial vinegar, while others, like the U.S. and Canada, allow raw vinegar sales provided safety requirements are met.

Final Exam Question

Question:

Which statement best describes the two-step fermentation process in apple cider vinegar (ACV) production?

A. Both alcoholic and acetic acid fermentation occur in the presence of oxygen, using Acetobacter bacteria to convert sugars into ethanol and then into acetic acid.

B. Alcoholic fermentation is anaerobic, where yeasts convert sugars into ethanol and carbon dioxide; acetic acid fermentation is aerobic, where Acetobacteria converts ethanol into acetic acid.

C. Alcoholic fermentation is aerobic, using yeasts to produce acetic acid directly from sugars; acetic acid fermentation is anaerobic, converting ethanol to acetic acid.

D. Both alcoholic and acetic acid fermentation require heat to accelerate the reaction and produce vinegar within a few days.

Correct Answer: B

Explanation for the correct answer:

Step 1: Alcoholic Fermentation (Anaerobic)

  • Process: Yeasts convert sugars in crushed apple juice into ethanol (alcohol) and carbon dioxide.
  • Oxygen condition: Anaerobic, meaning it occurs without oxygen.
  • Chemical equation: C6​H12​O6 ​→ 2C2​H5​OH + 2CO2​
  • This step turns sweet apple juice into hard cider.

Step 2: Acetic Acid Fermentation (Aerobic)

  • Process: Acetobacter bacteria convert the ethanol from step 1 into acetic acid (the main acid in vinegar).
  • Oxygen condition: Aerobic, meaning it requires oxygen.
  • Chemical equation: C2​H5​OH + O2​ → CH3​COOH + H2​O

Why the other choices are wrong:

A: Incorrect because alcoholic fermentation is an anaerobic process and does not occur in the presence of oxygen, and sugars aren’t directly converted by Acetobacter.

C: Incorrect because alcoholic fermentation is not aerobic, and yeasts don’t produce acetic acid directly from sugars.

D: Incorrect because heat isn’t a defining factor in these fermentations; the process takes longer and relies mainly on microorganisms and oxygen availability, not rapid heating.

References

  1. ↑ Apple cider vinegar. (2010). In C. E. Ulbricht (Ed.), Natural Standard herb & supplement guide: An evidence-based reference (1st ed., p. 59). Elsevier.
  2. ↑ 2.0 2.1 Rose, V. (2006). Apple cider vinegar: History and folklore—composition—medical research—medicinal, cosmetic, and household uses—commercial and home production. iUniverse.
  3. ↑ 3.0 3.1 3.2 Ousaaid, D., Laaroussi, H., Mechchate, H., Bakour, M., El Ghouizi, A., Mothana, R. A., Noman, O., Es-safi, I., Lyoussi, B., & El Arabi, I. (2022). The nutritional and antioxidant potential of artisanal and industrial apple vinegars and their ability to inhibit key enzymes related to type 2 diabetes in vitro. Molecules, 27(2), 567. https://doi.org/10.3390/molecules27020567 PMC
  4. ↑ Lea, A. (2022, August 24). How to make cider vinegar. Lowimpact. https://www.lowimpact.org/posts/how-to-make-cider-vinegar/
  5. ↑ Lea, A. (n.d.). The science of cidermaking: Part 6—Apple juice and cider vinegar. Wittenham Hill Cider. Retrieved August 9, 2025, from http://www.cider.org.uk/part6.htm.
  6. ↑ 6.0 6.1 Song, J., Zhang, J.-H., Kang, S.-J., Zhang, H.-Y., Yuan, J., Zeng, C.-Z., Zhang, F., & Huang, Y.-L. (2019). Analysis of microbial diversity in apple vinegar fermentation process through 16S rDNA sequencing. Food Science & Nutrition, 7(4), 1230–1238. https://doi.org/10.1002/fsn3.944
  7. ↑ 7.0 7.1 Ge, Y., Wu, Y., Aihaiti, A., Wang, L., Wang, Y., Xing, J., Zhu, M., & Hong, J. (2025). The metabolic pathways of yeast and acetic acid bacteria during fruit vinegar fermentation and their influence on flavor development. Microorganisms, 13(3), 477. https://doi.org/10.3390/microorganisms13030477
  8. ↑ Gomes, R. J., Borges, M. de F., Rosa, M. de F., Castro-Gómez, R. J. H., & Spinosa, W. A. (2018). Acetic acid bacteria in the food industry: Systematics, characteristics and applications. Food Technology and Biotechnology, 56(2), 139–151. https://doi.org/10.17113/ftb.56.02.18.5593
  9. ↑ Yarış, A., & Ceyhun Sezgin, A. E. (2017). Food packaging: Glass and plastic. In H. Araplıoğlu, A. Atik, R. L. Elliott, & E. Turgeon (Eds.), Researches on Science and Art in 21st Century Turkey (pp. 735–740). Ankara: Gece Kitaplığı. https://www.researchgate.net/publication/338554897_FOOD_PACKAGING_GLASS_AND_PLASTIC
  10. ↑ Denenberg, Z. (2023, January 11). Does apple cider vinegar go bad? Bon Appétit. https://www.bonappetit.com/story/does-apple-cider-vinegar-go-bad Bon Appétit
  11. ↑ Eden Foods. (n.d.). Amber glass protected. Eden Foods. Retrieved August 9, 2025, from https://www.edenfoods.com/articles/view.php?articles_id=181 edenfoods.com
  12. ↑ Schildhouse, J. (2024, September 25). Does vinegar go bad? Southern Living. https://www.southernliving.com/does-vinegar-go-bad-7486932
  13. ↑ 13.0 13.1 Pourmozaffar, S., Hajimoradloo, A., Paknejad, H., & Rameshi, H. (2019). Effect of dietary supplementation with apple cider vinegar and propionic acid on hemolymph chemistry, intestinal microbiota and histological structure of hepatopancreas in white shrimp, Litopenaeus vannamei. Fish & Shellfish Immunology, 86, 900–905. https://www.sciencedirect.com/science/article/abs/pii/S1050464818308180
  14. ↑ 14.0 14.1 Hlebowicz, J., Darwiche, G., Björgell, O., & Almér, L. (2007). Effect of apple cider vinegar on delayed gastric emptying in patients with type 1 diabetes mellitus: a pilot study. BMC Gastroenterology, 7(1). https://doi.org/10.1186/1471-230x-7-46
  15. ↑ 15.0 15.1 Yagnik, D., Serafin, V., & Shah, A. J. (2018). Antimicrobial activity of apple cider vinegar against Escherichia coli, Staphylococcus aureus and Candida albicans; downregulating cytokine and microbial protein expression. Scientific Reports, 8(1). https://doi.org/10.1038/s41598-017-18618-x
  16. ↑ Li, Y., Luo, Y., Lu, Z., Dong, Y., Chai, L., Shi, J., Zhang, X., & Xu, Z. (2023). Metabolomic analysis of the effects of a mixed culture of Saccharomyces cerevisiae and Lactiplantibacillus plantarum on the physicochemical and quality characteristics of apple cider vinegar. Frontiers in Nutrition, 10. https://doi.org/10.3389/fnut.2023.1142517
  17. ↑ Maladkar, S. R., Yadav, P., Muniraja, A. N. A., Uchil, G. S., George, L. V., Augustine, D., Rao, R. S., Patil, S., Sowmya, S. V., & Haragannavar, V. C. (2022). Erosive effect of acidic beverages and dietary preservatives on extracted human Teeth—An in vitro analysis. European Journal of Dentistry, 16(04), 919–929. https://doi.org/10.1055/s-0041-1742131
  18. ↑ 18.0 18.1 Health Canada. (n.d.-a). Food and nutrition – Standards of identity and composition. https://www.canada.ca/en/health-canada/services/food-nutrition.html
  19. ↑ Canadian Food Inspection Agency. (n.d.-a). Acts and regulations enforced by the CFIA. https://inspection.canada.ca/en
  20. ↑ Food and Drugs Act, R.S.C., 1985, c. F-27. (n.d.). https://laws-lois.justice.gc.ca/eng/acts/F-27
  21. ↑ Canadian Food Inspection Agency. (n.d.-b). Food labelling for industry. https://inspection.canada.ca/food-labelling
  22. ↑ 22.0 22.1 Health Canada. (n.d.-b). Food safety and the Food and Drugs Act. https://www.canada.ca/en/health-canada/services/food-nutrition/food-safety.html
  23. ↑ Health Canada. (n.d.-c). Participation in the Codex Alimentarius Commission. https://www.fao.org/fao-who-codexalimentarius
  24. ↑ U.S. Food and Drug Administration. (n.d.). Vinegar: Identity and composition. https://www.fda.gov
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