Dairy Fermentations (cheese) https://www.youtube.com/watch?v=ayM68-4EioY (2:15 – 3:15)

We could talk all semester about: Fermented dairy products; Fermented vegetables; Fermented meats Cocoa, coffee, tea Beer and wine Cereal and starch products (bread) Legumes (Natto, tempeh) Cereal-legume mixtures (soy sauce)

Spoilage or beneficial? Process Beneficial (controlled) Detrimental (uncontrolled) Conversion of sugars to lactic acid Acid flavor in fermented meats and dairy products Sourness of fresh meats, pasteurized milk Malty flavor (3-methylbutanal) Malt powder Pasteurized milk Ropiness (exopolysaccharides) Yogurt Meats, milk Acetaldehyde (product of threonine catabolism) Yogurt (green apple flavor) Milk Diacetyl Buttermilk (butter flavor) Fresh milk Again, discussing from standpoint of milk as this is best defined http://www.ncbi.nlm.nih.gov/pubmed/10553287; Lb. perolens produces high levels of diacetyl.

General Cheese Making Steps Raw Milk Heat Pasteurize/Heat Starter culture Inoculate with Starter and Ripen Add Rennet and Form Coagulum Cut Curd & Cook Low aw Drain Whey Acid development Texture Curd Dry Salt/Brine Salt (4%) Form Cheeses Store and Age

What is role of microbial metabolism in flavor development? Qualifier, this is a terribly complex topic and there is much more going on than I have time to tell you Fermentation isn’t just about preservation, but everytime you eat cheese you should be thanking microbes

The organisms - starters Product Primary acid producers Secondary microflora Colby, Cheddar, cottage Lactococcus lactis Lactobacillus adjuncts Gouda, Edam, Havarti L. lactis Citrate fermenting Leuconostoc or L. lactis Brick, Limburger Geotrichum candidum, Brevibacterium linens, Micrococcus, Arthrobacter Mozzarella, Provolone, Romano, Parmesan Streptococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus Lactobacillus adjuncts, animal lipases (Romano and Provolone) Swiss Streptococcus thermophilus, Lb. delbrueckii Propionibacterium freudenreichii subsp. shermanii Yogurt S. thermophilus, Lb. delbrueckii subsp. bulgaricus None Point being: one big difference is the microorganisms used What happens when you add microorganisms? Lactose utilized (energy source, precursor to membrane lipids), as well as milk proteins (we’ll get to that)

Homofermentative lactose utilization Lactococcus lactis lactose ATP phospho-b-galactosidase ADP glucose galactose-6-P ATP ADP tagatose pathway glucose-6-P tagatose-6-P In order to discuss these microorganisms, need to talk about the ways they utilize lactose Embden-Meyerhoff pathway fructose-6-P tagatose-1,6-diP fructose-1,6-diP 4 triose-P

Lactose  2 ATP + 4 lactic acid 4 triose-P 4 ADP 4 ATP 4 pyruvic acid 4 lactic acid Homofermentative = lactose makes one end product Recall that tricarboxylic acid cycle, one sugar molecule makes ~38 ATP; fermentation is rather inefficient process comparatively Lactose  2 ATP + 4 lactic acid

Ugly figure, focus on big picture Humans can’t make 9 amino acids; LAB used in dairy fermentations often can only make about this many Ugly figure, focus on big picture http://www.intechopen.com/books/milk-protein/bioactive-casein-phosphopeptides-in-dairy-products-as-nutraceuticals-for-functional-foods

What is happening during “aging”? Lactococcus lactis starter levels decrease Requires fermentable carbohydrate Becomes acid stressed Non-starter bacteria begin to grow Use other energy sources: citrate, amino acids, ribose and deoxyribose More acid-tolerant than L. lactis to pH conditions (approximately 5.1) Video from Italy describing where non-starters come from. A) Cheese making is not a sterile process especially in smaller facilities; B) Your plant

Organisms – non starters Called NSLAB, as most non-starters that contribute to flavor development are lactobacilli (acid tolerant, use many of substrates left in cheese) http://dairychemistry.blogspot.com/

Positive flavor impacts Acid Primarily lactic acid from starter Amino acids alone Proline = sweet Glutamic acid = meaty/brothy Amino acid catabolism Met  methanethiol; Cys  H2S Amino acids plus carbonyls Ever wonder why, in general, raw milk cheeses have a more complex flavor? One reason is additional microflora (also pasteurization doesn’t inactivate all enzymes, doesn’t boil off feed flavors coming in with milk) Carbonyl = carbon double bonded to an oxygen

methylglyoxal glyoxal ethanal Griffiths et al., J. Dairy Sci. 72:604-613

Defects Proteolytic-mediated bitterness Gas slits Fruity flavors; ethanol production by non-starters, esterification with short chain fatty acids Amino acid catabolism Phe  benzaldehyde Tryptophan  skatole/indole Tyrosine  phenol Benzaldehyde = almond Phenol = mediciny likely

Important to point out that milk caseins aren’t completely degraded (if they were, we’d have cheese soup!) Bitter peptides are those left behind http://pubs.rsc.org/en/content/articlehtml/2006/sm/b605670a

Heterofermentative lactose utilization Some lactobacilli lactose b-galactosidase glucose galactose 2 glucose-6-P ATP ATP ADP ADP

2 glucose-6-P 2 CO2 ADP ATP 2 xyulose-5-P 2 triose-P 2 acetyl phosphate acetate 2 pyruvic acid acetaldehyde 2 lactic acid ethanol

Gas slits in cheese Citrate acetate Oxaloacetate CO2 malate pyruvate fumarate diacetyl succinate

Other microflora Adjuncts (Lactobacillus helveticus for Cougar Gold) Can also use this to answer question: why are raw milk cheeses so much more flavorful than pasteurized? http://blog.bedfordcheeseshop.com/un-can-ny/

Microbiological cheese controversies 60 day aging process for raw milk cheeses 7 CFR 58 Part 7 is all about agriculture; 58 specifically is grading and inspection of dairy plants; the microbial standards for raw and pasteurized milk are found in 7 CFR 58 as well.

Aging on wooden shelves 21 CFR 110 Not so much a controversy as a hot button issue