Notes on Lactic Acid Fermentation and Pickle Fermentation

Lactic Acid Fermentation: Required Reading Overview

Introduction to Lactic Acid Fermentation

  • Lactic Acid Bacteria (LAB) convert carbohydrate contents (simple sugars) of vegetables and fruits into lactic acid (LA).

  • This process decreases the pH to approximately 4 or lower, ensuring product stability.

  • Benefits include enhanced safety and probiotic characteristics, as many LAB strains are known to have intestinal benefits.

Lactic Acid Bacteria (LAB)

  • Overview: A highly important taxonomic group of bacteria in food fermentations.

    • Distinguished by their production of lactic acid and genetic relatedness.

    • Note: Lactic acid production is not exclusive to LAB; other bacteria (e.g., Bacillus strains, Actinobacteria) also produce it.

  • General Characteristics:

    • Gram-positive.

    • Non-sporulating.

    • Comprises both bacillus (rod-shaped) and cocci (spherical) forms.

  • DNA-based Classification (Low GC Content):

    • LAB typically have low Guanine-Cytosine (GC) content or high Adenine-Thymine (AT) content in their DNA.

    • This characteristic was measurable long before DNA sequencing. Guanine and Cytosine form three hydrogen bonds, while Adenine and Thymine form two.

    • High GC content DNA requires more energy (heat) to denature (unwind) its strands because of stronger bonding. Low GC content requires less energy.

    • This measurable denaturing energy allowed for early classification based on DNA composition.

  • Anaerobic and Aerotolerant:

    • Most LAB are obligate fermenters, meaning they produce energy through fermentation.

    • However, they are also aerotolerant, having evolved mechanisms to protect themselves from the oxidative effects of oxygen.

    • Unlike many obligate anaerobes that die upon exposure to air, LAB possess protective mechanisms, though not all comprehensive ones (e.g., many are catalase-negative, lacking the enzyme to break down hydrogen peroxide).

    • Oxygen, while essential for many life forms, is a dangerous element for cells due to the free radicals it can generate, which are cell killers. Organisms like humans have co-evolved protection mechanisms.

Homofermentative vs. Heterofermentative LAB

  • Homofermentative: Produce only lactic acid as their main fermentation product.

    • Examples: Some Pediococcus, Streptococcus, and Lactococcus genera members.

    • Important Note: Homo- or heterofermentative designation is strain-specific, not genus or species-specific. For example, Lactococcus lactis has both homofermentative and heterofermentative strains.

  • Heterofermentative: Produce lactic acid along with other fermentation products.

    • Examples: Ethanol, Carbon Dioxide (CO_2), acetic acid, and other organic acids.

    • Also a strain-specific designation.

L-Lactic Acid vs. D-Lactic Acid Isomers

  • L-isomer:

    • Absorbed by the intestinal mucus.

    • Utilized as an energy substrate for metabolic activity in humans.

  • D-isomer:

    • Not assimilated by the body.

    • Eliminated by the kidneys in salt forms, potentially leading to calcium loss.

  • Prevalence:

    • Homemade or small-scale spontaneous fermentations often contain both L- and D-lactic acid isomers.

    • Commercial controlled fermentations aim to use starter cultures that produce only L-lactic acid.

    • Generally, L-isomer production is more prevalent even in natural fermentations; small amounts of D-isomer are usually not problematic for most people. However, some individuals with specific conditions can experience serious issues with D-llactic acid.

Bacteriocins

  • Definition: Antimicrobial peptides (AMPs) produced by bacteria.

  • Significance: Cultures producing bacteriocins contribute to greater safety in fermented foods by inhibiting undesirable microbes.

Principles of Vegetable Fermentation

  • Salting Methods:

    • Dry salting: (e.g., sauerkraut)

    • Brine salting: (e.g., pickles)

  • Non-Commercial Fermentations: Traditional methods (e.g., cassava) in indigenous settings, often relying on spontaneous fermentation to exclude oxygen. Early humans learned optimal conditions (e.g., salt) through experience to prevent pathogen growth.

  • Spontaneous vs. Controlled Fermentation:

    • Spontaneous: Relies on naturally present microorganisms.

    • Controlled: Involves introducing specific starter cultures for predictable and consistent results.

  • Auto- and Alloochthonous Cultures:

    • Autochthonous: Starter cultures isolated and characterized from the specific food product they are intended to ferment (e.g., Leuconostoc mesenteroides from sauerkraut used for more sauerkraut).

    • Allochthonous: Starter cultures isolated from a different source (e.g., soil, different fermentation) and then characterized for use in a specific food.

    • (These terms are generally considered less useful descriptors).

Factors Affecting Lactic Acid Fermentations

  • pH Optimality: Each culture has an optimal pH range for growth and activity.

  • Oxygen (O_2) Availability: LAB are aerotolerant but not strictly oxidative. While they have protection mechanisms, many are catalase-negative.

  • Temperature Optimality: Crucial for optimal growth and fermentation rates.

  • Salt Tolerance: Varies significantly among different LAB species and strains.

  • Water Activity (a_w):

    • Refers to the amount of water available for biological functions and metabolism.

    • Low water activity (due to drying or high solute concentrations like sugar or salt) inhibits microbial growth.

    • Yeasts and molds are generally more tolerant to lower water activity than bacteria, often explaining their growth in less ideal conditions.

  • Source of Nutrients: Availability of fermentable carbohydrates and other essential nutrients.

Selection Criteria for Commercial Starter Cultures

  • In a commercial setting, selected starter cultures are preferred for consistency and control.

  • Key Characteristics:

    • Non-toxicity: Absence of harmful compounds.

    • L-Lactic Acid Production: Exclusive production of the L-isomer to avoid issues with D-lactic acid.

    • Biogenic Amine Production (Low/None):

      • Biogenic amines are produced from amino acids through decarboxylation (e.g., Histamine from Histidine, Tyramine from Tyrosine).

      • Some, like Histamine and Tyramine, can cause sensitivities or problems in individuals with lower amounts of amine-breaking enzymes or those on certain medications.

      • Beneficial biogenic amines exist (e.g., Dopamine from Tyrosine metabolism, a "feel-good" hormone).

      • Genomic characterization helps identify an organism's capability to produce specific biogenic amines.

    • Genetic Stability: Ensures consistent performance over time.

    • Rapid Action/Functional Adenosine: Cultures that work quickly.

    • Reproducibility: Consistent fermentation outcomes.

    • Resistance: To bacteriocins (produced by other bacteria) and bacteriophages (viruses that infect bacteria, a common cause of fermentation failure).

Pickle Fermentation (Cucumbers)

Overview

  • Fermented cucumbers are a major commercial food fermentation product (e.g., dill pickles, sliced pickles).

Classes of Cucumber Products

It's crucial to distinguish between true fermented pickles and other pickled cucumber products:

  1. Fresh Pack Pickles:

    • Cucumbers are cleaned, placed in containers with a vinegar (acidic) solution, vacuumed, and heated (cooked).

    • These are shelf-stable and found at room temperature in stores. They are not fermented.

  2. Refrigerated Pickles:

    • Fresh cucumbers placed in containers with a seasoned (acidic, often sweet) solution.

    • The key difference from fresh pack is they skip the heating stage.

    • Require refrigeration for stability. Also not fermented.

  3. Fermented Pickles:

    • Cucumbers are cleaned and submerged in a brine solution in large tanks.

    • Undergo a long, cold fermentation process, sometimes for 1 to 3 months.

    • Microbial action naturally drops the pH to low 3s.

    • Yields a significantly richer and more complex flavor profile compared to non-fermented varieties.

    • More costly due to extended processing time.

Pickle Fermentation Process

  • Brining:

    • Cucumbers are placed in a salt solution.

    • Salt concentration is measured using a salometer (hydrometer).

    • Typical commercial pickle brines range from 20 to 30^ ext{o} salometer.

      • Note: 1^ ext{o} salometer is approximately 0.25^ ext{o}/o salt. Therefore, pickle brines are significantly higher than sauerkraut ( ext{approx.} 2.5^ ext{o}/o salt), usually >6^ ext{o}/_o salt.

    • The high salt concentration is critical as it influences the succession of microbial cultures, favoring salt-tolerant species.

  • Fermentation Type (Natural vs. Controlled):

    • Natural (Spontaneous): Relies on indigenous microbes. Less common in large commercial operations due to variability.

    • Controlled: Utilizes specific starter cultures, providing better consistency and control over the fermentation process.

  • Microbial Succession:

    • The high salt content in pickle brines discourages the growth of Leuconostoc mesenteroides.

    • L. mesenteroides is a heterofermentative LAB that produces CO_2 gas, which is undesirable in pickles as it causes bloater formation.

    • Instead, Pediococcus species and various Lactobacillus species are encouraged, as they are typically more salt-tolerant and homofermentative.

  • Industrial Setup:

    • Fermentation often occurs in large outdoor tanks, exposed to the environment (e.g., rain, birds).

    • The acidic environment created by LAB effectively kills off potential pathogens from environmental exposure.

    • Nitrogen purging is often used to remove air from the tanks, maintaining anaerobic conditions crucial for fermentation.

  • Completion:

    • Fermentation is complete when gas production (burping) ceases and the pH stabilizes, typically in the low 3s.

    • This process commonly takes several months.

  • Practical Consideration (Floating Cucumbers):

    • Cucumbers, like apples, float in liquid.

    • To ensure uniform fermentation and prevent exposure to oxygen at the surface, farmers use large weighted covers with holes (pallets) to keep the cucumbers submerged in the brine solution at the bottom of the tanks.

Spoilage Problems in Pickle Fermentation

  • Bloater Formation:

    • Characterized by blisters or hollowness within the pickles.

    • Caused by excessive CO_2 gas production by heterofermentative LAB (e.g., Leuconostoc mesenteroides).

    • Prevented by using homofermentative starter cultures and maintaining high salt concentrations to inhibit gas producers.

  • Loss of Texture ("Saggy Pickles"):

    • This is a major challenge in the industry.

    • Caused by pectinases, enzymes that break down pectin, a structural component of cucumbers.

    • Pectinases can be produced by various organisms.

    • Problem of Brine Recycling: Disposing of high-salt brine is expensive and ecologically harmful. Therefore, the industry recycles brine. However, repeated use leads to a buildup of pectinase enzymes, which can degrade texture.

    • Heat treatments are often used to try and manage pectinases in recycled brine, but complete and efficient removal remains a significant challenge.

  • Unclean Flavors and Odors:

    • Result from the growth of undesirable bacteria (e.g., Clostridia) or other spoilage microorganisms.

    • Controlled fermentations reduce the likelihood of these off-flavors.

Other Brine Fermentations

  • Products like pickle peppers and other whole fruit/vegetable fermentations utilize very similar brine-based processes.