26Fermentation

Fermentation is food changed by microbes: bacteria, yeasts and molds. They feed on the sugars in milk, cabbage, fruit or grain, and what they leave behind becomes yogurt, sauerkraut, vinegar, wine, beer and cheese. In science, the word usually means feeding that happens without using oxygen.

People have relied on it for a very long time. Cheese making has been traced to Mesopotamia, in the period 7000–6000 B.C.E., about the time beer was first brewed. Cheese is, at heart, a way to keep extra milk from going to waste. The acid that many of these microbes make is also why fermented foods keep longer than the fresh food they came from.

26.1 Microbes and how they feed

Two kinds of microbe

The microbes that make food fall into two big groups. Scientists call bacteria prokaryotes. Yeasts and other fungi, such as molds, belong to the second group, the eukaryotes. Each microbe used in food belongs to one group or the other. Among them are Saccharomyces, Lactobacillus, Leuconostoc, Streptococcus, Penicillium and Acetobacter.

Of these, Lactobacillus and Leuconostoc are bacteria that make lactic acid. Saccharomyces is a yeast.

Sugar: what microbes eat

Microbes run on carbohydrates, and the one they use most is glucose, a simple sugar. Every feeding route described below starts from glucose.

Sugars come in three sizes.

• Monosaccharide means "one sugar". These are the building blocks: glucose, fructose, galactose and others.
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• Disaccharide means two sugars joined. Maltose, the sugar of grain, is two glucose units. Lactose, the sugar of milk, is galactose plus glucose. Sucrose, table sugar, is glucose plus fructose.
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• Polysaccharide means a long chain of hundreds or thousands of sugar units. Starch in wheat, rice and barley is a mix of two of them: amylose, a straight chain, and amylopectin, a branched one. Cellulose, the stiff material of plant walls, is another.
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A two-sugar or many-sugar molecule cannot be used whole. It must first be split into single sugars by a matching enzyme — a protein that drives one particular chemical change. Lactase splits lactose; maltase splits maltose; amylase cuts starch chains. This splitting matters in beer, cheese and yogurt. Mammals cannot digest cellulose at all unless helpful bacteria in the gut do it for them.

The grains that supply starch are taught with grains, pasta and noodles. see chapter 16: What a grain is

Turning sugar into energy

A cell burns glucose in stages. The first stage is glycolysis: a chain of enzyme steps that breaks one glucose into two smaller pieces called pyruvate. Along the way the cell gains ATP, the small molecule that carries energy around inside a cell, and NADH, a carrier loaded with spare electrons.

When oxygen is plentiful, pyruvate goes on to a second stage, the Krebs cycle (also called the citric acid or TCA cycle), which ends by turning the carbon into carbon dioxide. The NADH then passes its electrons down a chain of proteins to oxygen, which becomes water, and that flow makes much more ATP. Using oxygen this way is called aerobic respiration.

Life without oxygen

Many microbes cannot use oxygen like that. Some lack the oxygen; some lack the genes for the parts of the machinery. Their problem is the NADH. Glycolysis uses up a carrier called NAD+ and leaves NADH, and unless the cell can empty those carriers again, glycolysis stops. Fermentation is the cell's way of emptying them without oxygen.

• A facultative microbe can live either way. It uses oxygen when it has it, and ferments when it does not.
• An obligate anaerobe cannot use oxygen at all, even when it is present, because it is missing parts of the Krebs cycle or the electron chain. Many bacteria are like this.

Lactic acid fermentation

This earns the cell no extra ATP, so it is a poorer living than breathing oxygen. A facultative microbe will switch back to oxygen when there is enough. But the acid is a weapon. It makes the food sour, slows down other living things and drives out competing microbes. People noticed long ago that food fermented this way lasts longer.

There are two styles.

• Homolactic fermentation makes lactic acid and nothing else. Lactobacillus delbrueckii and Streptococcus thermophilus, the bacteria used for yogurt, work this way.
• Heterolactic fermentation takes a side route through the sugar (called the pentose phosphate pathway) and makes a mix of lactic acid and ethanol, the alcohol in drinks. Leuconostoc mesenteroides works this way, and it is used to sour cucumbers into pickles and cabbage into sauerkraut.

Acid is one of the six conditions that control the growth of bacteria. Bacteria do not grow in acid, which is why acidic foods such as vinegar and lemon juice serve as preservatives. The full list of what bacteria need is with the rest of food safety. see chapter 2: What bacteria need: FATTOM

26.2 Vinegar

Alcohol made sour

Vinegar is made by bacteria that turn alcohol into acetic acid, the sharp acid in vinegar. In 1862 Pasteur gave the first account of this, and saw that a living thing was doing the work.

The workers are the acetic acid bacteria, often shortened to AAB, of the genus Acetobacter and its relatives. They live where there is sugar, alcohol or acid: on fruit and flowers, and above all in fermented drinks. Given enough oxygen, they turn ethanol into acetic acid. Producers feed them apple cider, wine or fermented grain mash. The same bacteria help make cocoa powder and kombucha, and they can also spoil food.

Those feeds are already alcoholic, so vinegar begins where an alcohol fermentation ends. How yeast makes alcohol is explained under Yeast.

How the bacteria do it

The change happens in two quick steps. Ethanol becomes acetaldehyde, and acetaldehyde becomes acetic acid. Each step is run by its own enzyme, and both enzymes sit on the outer side of the cell's membrane rather than inside the cell.

Ethanol, acetaldehyde and acetic acid are all poisonous to living cells, yet AAB thrive among them, for several reasons.

• The work is done outside the cell, so the poisons need not enter it.
• In many species the two enzymes are joined, so acetaldehyde is never set loose.
• Pumps actively remove hydrogen ions from the cell.
• The cell's membrane and its proteins are built to stay stable in strong acid.

The electrons taken from the alcohol are passed to the oxygen-using chain inside the bacterium. That is why vinegar bacteria need air: they cannot work without oxygen.

The second growth

Some Acetobacter strains, once the alcohol runs low, start to feed on the acetic acid itself, breaking it down to carbon dioxide and water. In a vinegar batch the bacteria grow in two bursts: first while making acid from alcohol, then again while eating that acid. Vinegar makers try to stop the second burst, since it destroys the product.

Vinegar's acid is also why it keeps. As an acidic food, vinegar does not support bacteria and can be used to preserve other foods.

26.3 Fermented vegetables

Three words: fermented, acidified, pickled

Vegetables can be kept with acid in two ways, and the words for them differ.

• A fermented vegetable is soured by microbes that make acid. Extra acid may or may not be added too.
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• An acidified vegetable is soured by adding acid directly.
• Pickle covers either kind, though it most often means vegetables kept in acetic acid.
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Cucumbers, cabbage and olives are the fermented vegetables sold most widely, but many others are made. Choosing and preparing the vegetables themselves is covered with vegetables. see chapter 17: Buying, storing and preparing vegetables

Why cabbage turns more sour than cucumber

Fresh cabbage holds about 4–8% sugars that microbes can ferment: glucose, fructose and sucrose. Cucumbers hold far less. More sugar means more acid, so a cabbage ferment ends at a much lower pH (more acid) than a cucumber pickle, and sauerkraut tastes sourer.

Vegetables also carry tough carbohydrates that microbes cannot easily use, known together as fiber. Two are cellulose, long chains of glucose, and pectin, the material that holds plant cells together.

Salting and packing

Yeasts and many other microbes already live on the skin of raw vegetables, so nothing needs to be added to start a ferment.

• Shred the cabbage, or prepare another suitable vegetable, and pack it into a jar.
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• Add salt, either dry or as a brine (salt dissolved in water), so that the vegetable ends up completely covered by liquid.
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• Press down firmly. Pressure squeezes juice out of the cabbage, and that juice carries the sugars and nutrients the bacteria need.

The salt does three jobs:

• It holds back many of the organisms that would spoil the food.
• It breaks open the plant cells so their sugars flow out for the bacteria.
• It seasons the pickle.

Salt may also stop enzymes that break down pectin and cellulose, which helps a pickle stay firm.

Two stages of bacteria

A vegetable ferment is called wild, because it relies on the lactic acid bacteria already on the vegetable rather than on an added culture. At first many kinds of bacteria compete. As the lactic acid bacteria release acid, the pH falls and most of the others die. Some producers put acetic acid into the brine right at the beginning.

• First stage. Leuconostoc bacteria, which need no oxygen, ferment the sugars. This is a heterolactic fermentation, so it makes more than lactic acid. The vegetables must stay under the brine and away from air for the first week.
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• Second stage. The acid grows too strong for Leuconostoc, and it dies out. Lactobacillus, which copes better with acid, takes over and keeps fermenting what sugar is left, making lactic acid, until the pH is down to about 3.

Flavor

In sauerkraut, Leuconostoc mesenteroides turns glucose into lactic acid, acetic acid and carbon dioxide. It also turns some fructose into mannitol, a sugar alcohol that tastes sweet, feels cool in the mouth and can hide bitterness. Given time, the bacteria ferment the mannitol into lactic acid as well.

That timing changes the result. In one taste test, most people preferred a partly fermented European sauerkraut to fully fermented kraut. The European kind held more leftover sugar, more mannitol and more ethanol, probably from wine added afterwards. But leftover sugar has a cost in bulk storage: spoilage organisms grow on it and gas builds up. A fully fermented sauerkraut has no sugar left and needs no further processing.

A second change can follow, called malolactic fermentation. Many strains of Leuconostoc and Lactobacillus turn malic acid, which vegetables contain naturally, into lactic acid and carbon dioxide. The gas causes trouble.

• In cucumbers it makes bubbles inside, softens the pickle, and gives hollow pickles known as "bloaters and floaters".
• In large tanks of sauerkraut it can cause heaving: the kraut swells fast with trapped gas and the brine level rises.
• Yet malic acid tastes harsher than lactic acid, and too much of it lowers how sauerkraut is rated, so producers must decide how far to hold this change back.

Sauerkraut tastes mainly salty, sour and sulfurous. Sulfur aromas belong to the whole cabbage family, radishes, kale and broccoli included, and fresh cabbage is rich in the compound that enzymes break down into many of sauerkraut's sulfur flavors. A little sulfur flavor is good; too much is not. Sauerkraut spiced with caraway is milder and less sulfurous, because the seeds seem to remove the starting material for some of the key sulfur compounds.

After fermentation

Spices, wine and other ingredients can be added to finished pickles for flavor. Cucumbers taken out of brine storage may be soaked or rinsed to make them less salty and less sour. Many pickles and krauts are pasteurized in their glass jars before they are sold.

A safety rule for sealed foods

26.4 Yeast

What yeast is and what it eats

Yeasts are fungi made of a single cell, and they are found almost everywhere in nature. They can feed on many things, including alcohols, acids and amino acids, but they prefer sugar. They can use simple sugars (glucose, fructose, galactose, mannose), the double sugars maltose and sucrose, and even ethanol.

The yeasts used in food are facultative: they can breathe oxygen, ferment, or do both at once. Yeasts are always present in the air, and they need food and warmth to grow.

Alcohol and gas

Yeast empties its NADH carriers by a different route from the lactic acid bacteria. It turns pyruvate into ethanol in two steps.

• An enzyme cuts a piece off pyruvate. That piece leaves as carbon dioxide gas, and what remains is acetaldehyde.
• A second enzyme adds electrons from NADH to acetaldehyde, which becomes ethanol. The emptied carrier goes back to glycolysis.

Every glucose a yeast ferments therefore gives off both alcohol and gas. Saccharomyces and other fermenting yeasts work this way. The kitchen uses both products. The gas is what makes bread dough rise. The alcohol, made on purpose, gives wine and beer. Made by accident, the same process makes fruit and vegetables ferment and rot. Food with unwanted yeast in it often turns slimy.

Why wine stops at about 16%

With oxygen or without?

Pasteur thought that yeast makes alcohol only when it runs out of oxygen, as a kind of starvation. That idea has since been shown to be wrong.

What actually happens is called the Crabtree effect. Breathing oxygen gives a yeast more energy from each sugar than fermenting does. Even so, when Saccharomyces finds itself in a lot of sugar, it starts making alcohol at once, while still breathing oxygen at the same time. It does this in beer wort (the sugary liquid that becomes beer) and in bread dough, however much air they hold. One idea is that the yeast makes alcohol to kill rivals that compete for the same sugar.

In short, Saccharomyces cerevisiae ferments when sugar is high, even with plenty of oxygen; breathes oxygen when sugar is low and oxygen is plentiful; and ferments when there is no oxygen. It can stand alcohol that most of its rivals cannot.

26.5 Cheese, yogurt and aging

The kinds of cheese and yogurt, and how to buy and use them, are covered with dairy. see chapter 14: Kinds of cheese

How acid and rennet set milk protein is covered with the other proteins. see chapter 7: Milk and soy

Where they came from

There is a story that desert nomads first made cheese by carrying milk in bags made from goat stomachs, where the rennet inside curdled it. Plausible as it sounds, the story is a myth. A similar story is told of yogurt: herders kept milk in bags made of animal gut, enzymes from the gut soured and thickened it, and the soured milk kept longer.

Cheese making goes back to Mesopotamia, around 8,000 years ago. By about 2000 B.C.E. most milk was drunk fermented, as something like a thin yogurt. Land that suited goats and sheep did not suit cows, so regions grew their own styles. In medieval Europe, religious houses made cheese both to eat in place of meat and to sell.

What is in milk

Milk is mostly water, with four main components:

• the sugar lactose
• fat
• the protein casein
• whey proteins

Casein floats in milk as tiny clusters, each made of hundreds of casein molecules held together with calcium. These clusters are called micelles.

Making cheese means drying milk down. Casein, fat and minerals end up 6 to 12 times as concentrated as in the milk, depending on the cheese. Most cheeses go through four steps:

• Making acid.
• Setting the curd.
• Removing water.
• Salting.

Three ways to set a curd

The curd forms when the casein clusters are made to clump together. There are three ways to do it.

• Rennet. Rennet holds an enzyme, chymosin, that snips one of the casein proteins, kappa-casein, so that part of it drifts away from the cluster. Without it, the clusters stop pushing each other away and stick together, with calcium acting as a bridge between them. A gel forms, the curd firms, and liquid seeps out. Cheddar and gouda depend on this.
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• Acid. Acid also makes the clusters clump. Lactic acid bacteria can supply that acid naturally. Cottage cheese, quark and cream cheese are made this way. An acid curd is more fragile than a rennet curd, because it loses its calcium.
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• Heat and acid. Heat changes the shape of the whey proteins so they join the casein, and then acid brings both out of the liquid together. Up to 90% of the protein is recovered. Paneer, ricotta and queso blanco are made this way.
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Whey proteins do not clump with rennet or acid alone, so they mostly drain away with the liquid.

In the kitchen, ricotta, mascarpone and crème fraîche (a thick soured cream) are set with acid at high heat and need no bacterial culture. They are known as "quick" or "kitchen cheeses".

Rennet is found in the stomachs of ruminant animals. It is sold as liquid, powder or tablets, and there are plant, animal and synthetic kinds. Liquid rennet from calves must be kept refrigerated; farmer's cheese and cheddar can both be set with it.

Raw milk or pasteurized

Raw milk is milk straight from the animal that has not been pasteurized. In the United States, some states allow raw milk products and some do not, so check the rules where you work. Raw milk is much richer in flavor and creaminess. Use it at once, ideally still warm; U.S.D.A. guidelines require raw milk to be processed within three days of milking.

Milk, and anything containing it, is a potentially hazardous food. The general rule for raw foods:

• Many carry pathogens.
• You cannot tell by look, taste or smell.
• Use processed or pasteurized versions wherever possible.
• Never serve such foods to high-risk customers.

A small cheese kitchen

Enough equipment to make at least six cheeses costs around five hundred dollars to start. Much of it is already in a working kitchen.

• A double boiler, or a saucepan set in a stockpot, and an accurate thermometer.
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• A curd knife; a flat cake-icing spatula does the job.
• Cheesecloth for straining, and plastic draining mats; sushi mats work.
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• Stainless steel bowls and ladles, and plastic tubs with lids for ripening cheese in the cold room.
• Cultures and molds to suit the recipe, such as a mesophilic culture, Penicillium candidum and Roqueforti.
• Calcium chloride, which makes the curd set more firmly in milk that has lost some strength to pasteurization.
• Rennet, and kosher salt or cheese salt with no anti-caking agent and no iodine.
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• Cheese molds, baskets, and presses or weights to drain off more whey and give the cheese its shape.
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From milk to curd

• Add the culture. Sprinkle the bacterial culture over the milk at the temperature the recipe gives, let it soak in for a few minutes, then stir gently with up-and-down strokes. Adding the culture is called inoculation.
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• Let it ripen. Leave the milk to rest while the bacteria turn lactose into acid.
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• Add rennet, then leave the milk undisturbed while the casein binds together. That binding is called flocculation.
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• Test for a clean break. Push a finger straight down into the set milk and lift it out sideways. When the curd splits cleanly over both sides of your finger, it is ready to cut.
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• Cut the curd, first with vertical strokes through the whole batch, then horizontal ones, to the size the recipe gives. Let the curds rest five minutes. Cutting speeds up the souring.

Curd size sets how moist the cheese will be: bigger pieces hold more whey. Some curds set by lactic acid alone, such as camembert's, are too delicate to cut, so they are ladled straight into baskets to drain.

After resting, the curds usually sink and a yellowish whey rises. Rennet curds are often cooked in their whey until they mat into a cake at the bottom of the pan, as for cheddar; other cheeses simply sit in the whey at a set temperature. Depending on the style, the curd may then be milled (broken or cut into pieces), salted, and put into hoops or pressed to squeeze out more whey. The cheeses are then turned on a schedule, and some are salted again whole.

Some cheeses, such as Colby and Gouda, have their curds washed, which leaves them moister and less acidic. Salt goes in different ways: Gouda is soaked in brine, while feta is salted on the surface.

Ripening: where flavor comes from

Flavor sources in ripening cheese.

The breakdown of fat, called lipolysis, is a key step. It splits fats into smaller acids and related compounds that are strong in both smell and flavor.

In Swiss cheese, bacteria called Propionibacterium ferment sugar into propionic acid, which gives the cheese its typical aroma and flavor. These bacteria are facultative: they breathe oxygen when they have it and ferment when they do not.

Lactic acid protects a cheese as well as flavoring it. By lowering the pH it keeps out unwanted bacteria and molds, while other products of the bacterium Lactococcus lactis add the subtle aromas that set one cheese apart from another.

Camembert can be aged in a covered plastic "cheese cave" at 60ºF, which lets its white mold grow. Some cheeses are coated in wax for storage and aging.

Yogurt

In the United States, the FDA defines yogurt as milk fermented by two bacteria, Lactobacillus bulgaricus and Streptococcus thermophilus. Both make lactic acid from the lactose in milk.

Yogurt's tartness is usually put down to lactic acid, but several other compounds add to the tang.

• Acetaldehyde. Yogurt bacteria make most of it from threonine, an amino acid, which they split into acetaldehyde and glycine. Goat's milk has more glycine than cow's milk, and goat's milk yogurt holds much less acetaldehyde.
• Diacetyl, made by both yogurt bacteria, gives a distinct buttery taste, here and in other fermented milks.
• Acetoin, a close relative of diacetyl, adds a mild, creamy note.

Living yogurt cultures release lactase in the gut, which goes on breaking down the lactose in the yogurt; this makes yogurt edible for people who cannot digest lactose. To make it keep longer, makers often heat yogurt after fermenting it, which kills those live cultures.

The two yogurt bacteria are killed by stomach acid and digestive enzymes. So makers often add other strains, called probiotics — for example Lactobacillus acidophilus, Lactobacillus casei and Bifidobacterium — for which there is evidence of reaching the gut intact. Alongside them they may add prebiotics, such as inulin, which help those bacteria grow in the gut.

Kefir

Kefir is a fizzy fermented milk drink. It is started with kefir grains: a living mix of lactic acid bacteria and yeasts held together in a mass of protein, fat and complex carbohydrate.

The bacteria act first, turning lactose into lactic acid; the pH drops and the milk is preserved. Yeasts and acetic acid bacteria join in, and propionic acid bacteria break part of that lactic acid down further, to propionic acid, just as in Swiss cheese. In a second fermentation, yeasts that can eat lactose turn it into ethanol and carbon dioxide, which gives kefir its fizz. The alcohol stays low, usually 0.2–0.3%. Its flavor, like yogurt's, owes much to diacetyl, acetoin and acetaldehyde.

Storing them

Dairy products are stored at 2–4°C (36–39°F), covered, in their own part of the fridge, and rotated as new stock arrives. Mold grows on cheese kept warm, dark and a little damp, and dairy that is spoiling turns darker and smells sour.