Demystifying Fermented Foods What They Are and How They Contribute to Healthy Diets

Tuesday, September 10th 2019 1:00pm EST

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Maria L Marco, PhD Professor Department of Food Science & Technology University of California, Davis

Affiliation/financial interests (past 12 months) ▪ Grants/research support: USDA, NIH, CDRF, Innovation Funds, Denmark ▪ Scientific Advisory Boards: Kerry Health and Nutrition Institute, International Scientific Association for Probiotics and Prebiotics, International Probiotics Association, The Fermentation Institute, NIH Botanical Dietary Supplements Research Center in Pennington, LA Outline

▪ Introduction and definition of fermented foods

▪ Brief description of how fermented foods are made and their characteristics

▪ Microbial and metabolic factors involved during the making of fermented foods

▪ Fermented foods and health benefits: clinical and epidemiological evidence

▪ Microbial processes and products contributing to health-impacting properties of fermented foods History of Fermented Foods Modern Day Cuisine Definition

Fermented foods are foods or beverages made as a result of extensive microbial growth. Sugars Lactic acid bacteria Yeasts Prepare Incubate Chop Temperature Salt Oxygen Spice pH Pack Water activity Soak Heat Molds Proteins, fats, Lactic acid bacteria polysaccharides Yeast Others Thousands of Fermented Foods & Beverages

▪ lactic acid: kimchi, sauerkraut, yogurt, sausage, cheese ▪ alcoholic: wine, beer, spirits ▪ CO2 (gas) containing: bread, kombucha, sparkling wines ▪ acetic acid: vinegar, kombucha ▪ mold-modified: tempeh, soy sauce, cheese, sausage Additional Details on Fermented Foods

▪ Fermentations can be spontaneous (wild) or initiated using “starter cultures” or a small portion of a prior ferment (backslopping)

▪ They are made using single (example: yogurt) or multiple (example: soy sauce) steps and can take hours, days, weeks to months prepare

▪ Depending on the food, a pasteurization step (heating) can be included (example: sourdough bread)

▪ There are no standards of identity for most fermented foods Making Fermented Foods: Microbial and Metabolic Factors Microbial Community Dynamics During Production of Fermented Foods

Lactic acid bacteria (LAB) are the most abundant microbes in olive fermentations 8

3 6 Company 1 Early LAB grew on the olives to ~10 cells /g Company 1 Mid Log(CFU/g) Company 1 Late 2 Company 2 Early Company 2 Mid Company 2 Late 1 Company 3 Early Company 3 Mid Company 3 Late 0 0 30 60 90 120 150 180 210 240 270 Days Post Submersion

LAB were quantified on MRS medium containing Natamycin (25 ug/ml) avg ± stdev CFU/g of 5 replicates per processor are shown

Zaragoza, Bendiks et al Microbial Community Dynamics During Production of Fermented Foods

Yeast also contribute to olive fermentations

8 Company 1 Early Company 1 Mid Company 1 Late 7 yeast grew on the olives to ~1000 cells/g Company 2 Early Company 2 Mid Company 2 Late 6 Company 3 Early Company 3 Mid Company 3 Late 5

Log(CFU/g) 3

0 0 30 60 90 120 150 180 210 240 270 Days Post Submersion

Yeast were quantified on RBCA medium containing Chloramphenicol (100ug/ml) avg ± stdev CFU/g of 5 replicates per processor are shown

Zaragoza, Bendiks et al Investigating Food Microbiomes

Culturable bacteria High-throughput DNA sequencing Food Fermentations are Dynamic Ecosystems

Leuconostoc Pediococcus

Pichia

Lactobacillus Candida

Zaragoza, Bendiks et al Microbial Metabolism: Production of Flavor Compounds

Proteins molds, Bacillus amino acids (Clostridium) many peptides microbes amino acids, amines NH3 NH3

molds Fats (glycerol) molds fatty acids ketones (esters)

molds yeast Starch Bacillus short-chained esters polysaccharides mono/di-saccharides

lactic acid Sugars bacteria lactic acid, acetic acid, acetaldehyde, diacetyl, CO2, ethanol

yeast ethanol, CO2 Microbial Metabolic Networks: More Than Just Flavor

Filannino et al 2018 Microbial Metabolism: Causes and Consequences of Different End Products

Glu

▪ Lactic acid bacteria make organic acids which lower the pH of foods ▪ To maintain intracellular pH, LAB can use amino acids and malate and this results in the production of flavor and health-impacting compounds

Ganzle 2015 Microbial Metabolism: Symbiotic Interactions and Fermentation Outcomes

Yogurt fermentations

▪ Streptococcus thermophilus

▪ Lactobacillus delbrueckii subspecies bulgaricus

Sieuwerts et al 2008 Fermented Foods & Health Benefits: Clinical and Epidemiological Evidence Human Studies on Fermented Foods

Obesity Depression Kimchi, Chung kook jang, Coffee, fermented milk kochujang

Type 2 Diabetes Infection Yogurt, fermented milk, kimchi Fermented milk

Cardiovascular disease Fermented milk, fermented soy Inflammatory bowel syndrome Sourdough bread

Marco et al 2017 Yogurt

Made using Streptococcus thermophilus and Lactobacillus delbrueckii subspecies bulgaricus. Many yogurts contain other added strains. ▪ Reduced risk for T2D (observational; meta- analyses) ▪ Inverse association with adiposity factors (observational studies) ▪ Reduced risk for CVD (longitudinal)

DB: double blind CO: cross over RCT: randomized control trial Fernandez et al 2017 Buziau et al 2019 Kefir & Fermented Milk

Made using different lactic acid bacteria species and yeast (kefir). ▪ Kefir: Improvement in bone mineral density in osteoporosis patients (DB-RCT) ▪ Kefir: Improved Helicobacter pylori symptoms (DB-RCT) ▪Fermented milk: Reduction in muscle soreness (DB-RCT)

Tu et al 2015 Bekar et al 2011 Iawasa et al 2013 Sauerkraut & Kimchi

Sauerkraut is made using cabbage. Kimchi is made using cabbage, radishes, and other vegetables, fruits, and spices. Both are lactic fermentations (Leuconostoc; Lactobacillus). ▪ Sauerkraut: Reduced IBS severity scores (DB-RCT) ▪ Kimchi: improved insulin sensitivity, blood pressure, and metrics of adiposity (body weight, body mass) (DB-CO-RCT) ▪Kimchi: lower presence of atopic dermatitis (cross- sectional (KHANES)

Nielsen et al 2018 Kim et al 2011 An et al 2013 Sourdough Bread

Sourdough bread is made with the help of yeast (Candida milleri, Saccharomyces cerevisiae) and lactic acid bacteria (Lactobacillus sanfransiscensis) ▪ Better tolerated by IBS patients, possibly due to lower concentrations of non-digestible oligosaccharides (FODMAPS) (DB-RCT) ▪ Improved GI tolerance (bloating, gastric volume) in healthy subjects (DB-CO-RCT) ▪ Some reduction in glycemic responses (RCT)

Laatikainen et al 2016 Polese et al 2018 Korem et al 2017 Health-Impacting Properties of Fermented Foods How Microbes Change Foods

Preservation is enhanced

Safety is frequently improved

Transform food components

Synthesize new compounds

Increase numbers of living microbes

Marco et al 2017 Preservation

Fermented foods typically have longer shelf-life times

▪ Increased organic acids (lower pH) Captain Cook was awarded a medal ▪ Increased quantities of other by the Royal Society for stopping antimicrobial compounds scurvy with sauerkraut (1775)

▪ Reduced quantities of nutrients available for spoilage microbe growth

▪ This does not apply to all fermented foods (e.g. natto, soft cheeses) Safety

Certain fermentations are acidic (pH < 4.5), decreasing the risk for foodborne illness

▪ Lactic acid bacteria (LAB) and acetic acid bacteria (AAB) acidify foods with lactic acid, acetic acid, and other organic acids

Possible safety concerns: ▪ Fermented foods made using molds have a higher pH and therefore pose more of a safety risk (e.g. blue cheese, brie, tempeh)

▪ Other negative safety concerns for fermented foods include the production of biogenic amines Transform Food Components

Remove phytic acid (myoinositol Reduce or remove toxic hexaphosphate), an anti-nutrient in compounds and anti- cereal grains that blocks mineral bioavailability (e.g. Fe, Ca, Zn, and Mn) nutrients in foods

• Reduce lactose concentrations (in dairy products) Improve food • Remove sugars that cause flatulence (raffinose and digestibility stachyose) • “weaning foods” – (porridges after starch digestion) Synthesize New Compounds

• Bioactive organic acids • γ-Aminobutyric acid (GABA) Increase vitamin & bioactive • Conjugated linoleic acid (CLA) compound content in foods • Vitamins B9 (folic acid), • B12 (coalbumin), B2 (riboflavin), B1 (thiamine)

Produce Compounds that modulate the gut prebiotics microbiome to benefit human health Increase Numbers of Living Microbes

Some fermented foods can increase the number of microbes consumed in a day by 100 to 10,000-fold

“Live and Active Cultures” is a good term to apply to fermented foods Some Microbes in Fermented Foods Survive Digestive Tract Transit

David et al 2013 Living Microbes ≠ Probiotic

▪ Probiotics are living microbes that when consumed in adequate amounts confer a health benefit

▪ Because fermented foods typically contain undefined microbial strains that have not been shown to benefit health, those foods should not be considered probiotic

▪ Dead microbes, microbial products, microbial components also do not come under the probiotic classification

Hill et al 2014 But What is a Strain?

PHYLUM Firmicutes CLASS Bacilli ORDER Lactobacillales FAMILY Lactobacillaceae

GENUS Lactobacillus

SPECIES casei Shirota STRAIN DN-014-001 BL23

Strain definition: A population of cells descended from a single isolate Bacterial Strains are Highly Diverse

Lactobacillus plantarum strains 42 L. plantarum strains incubated for 24 h with human peripheral blood mononuclear cells (PBMCs)

2500

2000 Anti-inflammatory IL-10

1500 induced over an 8-fold range 10 (pg/ml) 10

- 1000 IL 70% genetically related* 500

0 Variation in PBMC 0 4 8 12 16 20 24 28 32 36 40 responses was greater 60 than found between 50 different genera 40

12 (pg/ml) 12

- IL 20 Pro-inflammatory IL-12 10 induced over a 16-fold range

0 78% genetically related 0 4 8 12 16 20 24 28 32 36 40 strain (random assignment)

Staley 1997 van Hemert et al 2010 Possible Overlap Between Probiotics and Microbes in Fermented Foods

▪ There can be genus and species overlap in effects on the human body

▪ Some Lactobacillus species are both used as probiotics and used to make fermented foods (Lactobacillus plantarum, Lactobacillus casei)

Hill et al 2014 Fermented Foods: Beyond Basic Nutrition

Modulate the gut microbiota ▪ prebiotics ▪ organic acids ▪ antimicrobial compounds Improve barrier function ▪ organic acids ▪ secreted bioactive proteins

Stimulate immune responses ▪ exposure to living microbes

Alter the gut-brain axis ▪ neurotransmitters (GABA)

Yang et al 2018 Key Takeaways

▪ Fermented foods and beverages are important components of human diets ▪ The unique textural and sensory qualities of fermented foods are a result of microbial growth and metabolism ▪ Health altering aspects of fermented foods are due to the ingredients in those foods combined with microbial growth and metabolism (transformation and synthesis) ▪ Just because a fermented food has living bacteria does not mean it is “probiotic” ▪ There are multiple ways in which microbial metabolites and living microbes in fermented foods might cause systemic effects via the digestive tract More Research is Needed

▪ Many more human studies are needed to evaluate the health altering capacities of individual fermented foods and fermented foods as a food category ▪ Molecular, mechanistic research is needed to identify and verify the specific molecules in fermented foods (and living microbes) responsible for altering intestinal and systemic health ▪ Outcomes: Identification of health-benefiting fermented food formulations and personalized recommendations for adequate daily intake thank you! Q&A