by Debra Wink
This article was first published in Bread Lines, a publication of The Bread
Bakers Guild of America. Vol. 16, Issue 2, June 2008.

Pineapple juice is a simple solution to a problem that many people encounter
while trying to start a sourdough seed culture from scratch. Oftentimes, a new
culture will appear to start off very strong, only to die a day or two later.
The early expansion is caused by a prolific gas-producing bacterium which many
mistake for yeast. Pineapple juice can be added to flour instead of water at
the beginning, to insure against unwanted bacteria and the problems they leave
in their wake. It doesn't change the end result, but it does seem to keep
things on the track to finish on time. Part 1 tells the story of where the
pineapple remedy comes from and how it was conceived. The rest of the story
probes deeper into how it all works. But first, here is a recap of the key
patterns revealed by notes and data collected during experimental trials:

€¢ When starters expanded significantly on the second day, a period of stillness
followed, and the appearance of yeast was delayed.
€¢ Gas-producing bacteria stopped growing when the pH dropped to 4.5, but yeast
growth didn't begin until the pH fell to around 3.5, accounting for the period
of stillness.
€¢ Lowering the pH in the initial mixture, by adding ascorbic acid or by
replacing the water with pineapple juice, kept gas-producing bacteria from
growing and brought about a more timely and predictable result.

But it wasn't enough just to find a fix. The problem-solving efforts of my team
were creating a buzz which we hadn't anticipated and this thing, like the seed
cultures we were creating, was taking on a life of its own. Some were jumping
to premature conclusions, and speculation seemed to be spreading as fact. It
made me very uncomfortable, because I'd rather be dispelling myths than adding
to them. I wanted to find some real answers, and find them fast, so I started
making phone calls. I found two local labs that could help me out. One had the
capability to identify leuconostocs, and the other to detect lactobacilli and
other bacteria of interest. I submitted samples of a day two starter during the
big expansion. Both labs found that there were three organisms growing. But
there were no lactobacilli or yeasts found, which supports what I observed time
after time on microscopic examination. My gas-producer was identified as
Leuconostoc citreum. At the time, I couldn't find much information specific to
this organism, although it seems to share many characteristics with other
Leuconostoc species found in foods. Most will not grow below pH 4.8, and this
one doesn't appear to be an exception.

Until recently, I could only theorize that the Leuconostoc may actively hinder
the process, because the pattern supports it, and because it's not uncommon for
microorganisms to produce substances which inhibit competitors. But in updating
this article, a new search of the scientific literature finally uncovered the
piece of the puzzle I was looking for. Who would have thought the answers would
be found in kimchi and sake? It turns out that kimchi fermentation has a lot in
common with sourdough development, and mirrors the early days of the seed
culture process. Leuconostoc citreum plays a dominant role in the early and
mid-phases of fermentation where it causes a slow and prolonged drop in pH, and
retards the growth of other lactic acid bacteria.[1] In a study on sake
fermentation, Leuconostoc citreum was found to produce bacteriocins
(bacterially-produced antibiotic proteins) which inhibit the growth of similar
lactic acid bacteria (i.e., lactobacilli).[2] It appears that these
bacteriocins linger for a time even after the organism stops growing, although
their effect is diluted through successive feeding. A dosage effect would
explain nicely the apparent relationship between the vigor with which this
bacterium flairs up initially, and the number of days the starter remains still
afterward. The higher the rise, the longer it seems to take to recover.

In addition to Leuconostoc citreum, there was also a large amount of Aerococcus
viridans. The first lab I visited found Leuconostoc to be in the greatest
quantity, but Aerococcus was multiplying so fast that it soon passed the
Leuconostoc in number. That is important, and could very well have contributed
to the delayed progress. Even though Aerococcus doesn't produce gas, and so was
not responsible for any of the expansion, it is not an acid producer either. So
while it was using up a large share of the available sugars, it was not helping
the pH to fall. Aerococcus is an occasional spoilage organism in unpasteurized
milk, which is the extent of information that I have found on its involvement
in foods. Its lower limit is not given in my reference books, but since
pineapple juice seems to keep it at bay, I suspect that it must be in the same
ballpark with leuconostocs. I'm still not sure how big a part each of these
organisms plays in slowing the progress of a seed culture, but lowering the pH
at the outset seems to be a blanket fix.

I mentioned in Part 1 that some of the bacteria were flipping, twirling and
zipping around under the microscope. Those were Enterobacter cloacae.
Enterobacter produces gas, but since it was present in only a scant amount
compared to the others, I think it safe to say that the Leuconostoc was
responsible for the majority of it. However, Enterobacter contributes to an
unpleasant odor, as do Aerococcus and Leuconostoc. Because some people report a
very stinky smell and others not as much, I'd have to say that even among
starters that grow Leuconostoc, not all necessarily have the same combination
of bacteria. There are others that can grow as well. Results vary from flour to
flour and year to year, because the number and species of microorganisms are
influenced by conditions relating to weather and grain crop production.[3] I
wish I could have all the organisms identified at every stage, but there aren't
any laboratories in my area that are equipped to identify wild yeasts or
sourdough bacteria. And even if they could, the cost would be prohibitive. I
was fortunate to be in a position to have two of the organisms identified as a
professional courtesy.

With the additional information, and having watched the drama unfold under the
microscope, I started seeing the seed culture process not as good guys
out-competing bad or gradually increasing in number, but as a natural
succession of microorganisms that pave the way for "the good guys" in the way
that they transform their environment. There are bacteria in flour that prefer
the more neutral pH of freshly mixed flour and water (like Leuconostoc and
company). They are the first to start growing, some producing acids as
by-products. This lowers the pH, and other bacteria begin to grow; they produce
their acids, lowering the pH even more. It soon becomes too acidic for the
first batch and they stop growing. One group slows down and drops out as the
next is picking up and taking off. Each has its time, and each lays the
groundwork for the next. It's much more like a relay than a microbial
free-for-all. The baton is passed to the next group in line as conditions
become suitable for them. The acidity increases a bit more with each pass, and
the more acid-loving bacteria can eventually take over. The appearance of yeast
seems to be tied in some way to low pH---maybe directly, maybe indirectly, but
the correlation shows that it isn't random in the way that "catching" yeast
from the air would be, or their gradually increasing in number.

In the late fall/early winter of 2004, I was coaching a group of women on Cooks
Talk, Taunton's Fine Cooking forum, and I noticed something else. My starters
sort of liquefy the day before yeast starts to grow. Gluten disappears, which
shows the work of proteolytic enzymes. At first I thought it signaled the
appearance of lactobacilli and their proteases. But now I think it was simply
an indicator that the pH had dropped low enough to activate aspartic
proteinase, a pH-sensitive enzyme abundant in wheat.[4] Because I prefer to
seed a new culture with whole grain flour for at least three days, there are
more cereal enzymes present than in a starter fed with white flour (most of
them are removed with bran in the milling process). But either way, it is a
good sign of Lactobacillus activity, whether by production of bacterial
proteases or by the organism's effect on pH and activation of cereal proteases.

The starters were developing a little more slowly this time around, which
inspired me to describe the different stages that a new culture transitions
through, rather than try and pin it to a time frame. Room temperature is
different from one kitchen to the next, as well as season to season. Sometimes
rye flour works faster, sometimes whole wheat is faster. Sometimes a culture
doesn't start producing its own acid for the first two days instead of one.
Because this process involves variable live cultures under variable conditions,
it doesn't always work in a prescribed number of days, but it follows a
predictable pattern. While this has been a discovery process for me, it is not
a new discovery:

"There has been nice work done in Rudi Vogel's lab on the microflora of a
freshly started sourdough: first, there are enterobacteria (Escherichia coli,
Salmonella, Enterobacter), highly undesirable organisms that stink terribly.
Then there are homofermentative lactobacilli (good lactic acid producers, but
they don't produce gas or acetic acid), then acid-tolerant, heterofermentative
lactobacilli that make lactic and acetic acid, as well as CO2. I think this
took about forty-eight hours at 30ºC in Vogel's study. The stink at the
beginning does not matter as the organisms will be diluted out or die
eventually. No L. sanfranciscensis appears by forty-eight hours, though: these
will occur only after repeated refreshments. Peter Stolz told me that it takes
about two weeks of repeated inoculations to get a good 'sanfranciscensis'
sourdough."[5]

That paragraph didn't have any special significance for me until I had gotten to
this point. But when I read it again, I had one of those aha moments. Not only
did this describe a succession, but it filled in some of the blanks, and I
could see clearly how all these microorganisms related to the four phases I had
defined. Here is the updated version marrying the two. You don't need a
microscope for this, because there are outward signs which serve as useful
indicators of progress.

The First Phase:
For the first day or so, nothing really happens that is detectable to the human
senses. It doesn't taste any tangier or develop bubbles. It remains looking
much the same as when it was mixed, except a little lighter in color if an acid
was used, and a little darker if not. While nothing appears to be happening,
the first wave of bacteria (determined by pH and the microflora in the flour)
are waking up, sensing their new environment and preparing to grow. This phase
usually lasts about one day, sometimes two.

The Second Phase:
The starter will begin producing its own acid and develop a tangy taste
(although it might be difficult to distinguish from pineapple juice). Lactic
acid bacteria are actively growing at this point. When using only water, this
phase represents two waves of microbes---first Leuconostoc and associates,
followed by homofermentative lactobacilli and possibly other lactic acid
bacteria. By controlling the pH, you can by-pass the leuconostocs and other
"highly undesirable organisms that stink terribly," and skip to the second
wave. It will get bubbly and expand only if the pH is not low enough to prevent
growth of gassy bacteria, otherwise there won't be much else to see. There
probably won't be much gluten degradation, and it may smell a little different,
but it shouldn't smell particularly foul unless started with plain water. This
phase can last one to three days or more. If it is going to get hung up
anywhere, this is the place it usually happens, especially if it is put on a
white flour diet too soon. If after three days in this phase, it still doesn't
become more sour and show signs of progress, the best thing to do is switch
back to whole grain flour for one or more feedings. Whole grain flour has a
much higher microbial count and will re-seed the culture and get it moving
again.

The Third Phase:
The starter will become very tart---an indication of more acid production by
more acid-tolerant bacteria. The gluten may disappear and tiny bubbles become
more noticeable. These are signs that heterofermentative lactobacilli have
picked up the baton. Once a starter becomes really sour, it usually transitions
right into phase four. Note that lactic acid doesn't have much, if any aroma,
and so smell is not a very reliable way to judge the level of sourness.

The Fourth Phase:
Yeast start to grow and populate the starter relatively quickly at this point.
It will expand with gas bubbles all over and begin to take on the yeasty smell
of bread or beer.

This pattern suggests that wild yeasts are activated by low pH. Or perhaps the
activator is something else produced by lactobacilli, but it happens
predictably at this point for me, as long as the whole grain flour has not been
diluted out. There may be some variation among wild yeasts as to the exact pH
or activating substance. I have been unable to find the answer in scientific
literature, and my contact at Lallemand did not know. I have only found studies
done with cultivated strains of Saccharomyces cerevisiae, which don't seem to
require much more than a fermentable sugar. The most useful information I have
found on the subject is this, about microbial spores in general:

"Although spores are metabolically dormant and can remain in this state for many
years, if given the proper stimulus they can return to active metabolism within
minutes through the process of spore germination. A spore population will often
initiate germination more rapidly and completely if activated prior to addition
of a germinant. However, the requirement for activation varies widely among
spores of different species. A number of agents cause spore activation,
including low pH and many chemicals... The initiation of spore germination in
different species can be triggered by a wide variety of compounds, including
nucleosides, amino acids, sugars, salts, DPA, and long-chain alkylamines,
although within a species the requirements are more specific. The precise
mechanism whereby these compounds trigger spore germination is not clear."[6]

What this means is that for dormant cells to return to active growth
(germinate), they need to break dormancy (activate) which is initiated by
different things for different species. In the case of these wild sourdough
yeasts, if all they needed were food or oxygen, which are there from the
get-go, then they would start growing immediately. The fact that they don't, is
probably why many people think they need to be caught from the air, or that
large quantities of flour must be used to round up enough of them. There are
enough dormant cells present even in relatively small quantities of whole grain
flour, but it's like a game of Simon Says. You can try to coax them into
growing, with food and all the things you may fancy to be good for actively
growing yeast. But they're not active. They are dormant, and will remain so
until they receive the right message from their surroundings. Compare this to
the plant seed that sits in soil all winter long, waiting until spring to
sprout, when conditions are most favorable. Is it a survival mechanism? I don't
know, but waiting for the pH to drop does increase the likelihood that the
yeast will wake up in the company of lactobacilli, with which they seem to
share a complex and mutually beneficial relationship. It is also important to
point out here that active sourdough yeasts thrive in a much wider pH range
than what appears to be required for activation of dormant cells. The point to
keep in mind is that active and dormant cells are physiologically and
metabolically different, which also means their needs are different.

This pattern of growth is not unique to the formula in the Bread Baker's
Apprentice. I have seen the same progression, in whole or in part, with all the
starter formulas I've tried. And it doesn't really matter how much flour you
start with. In fact this can be done with very small quantities of flour. All
else being equal, it proceeds just as fast with a teaspoon as it does with a
pound. Procedures that call for two or three feedings per day, or large
refreshments before yeast are active, can actually get in the way of the
process. Overfeeding unnecessarily dilutes the acid, which slows the drop in
pH, and keeps it from moving through the succession of microorganisms in the
timeliest manner. But while it can take up to two weeks or more this way, with
Mother Nature as the driving force, things do fall in line eventually. It's
just a question of when. Three to five days is about all it really takes to
reach the yeast activation stage at average room temperature, somewhat longer
if Leuconostoc and associates grow. The strategy is quite different from
reviving a neglected starter, which is likely to have an overabundance of acid,
and a large population of yeast and sourdough bacteria, however sluggish they
may be.

So, what can we do instead to facilitate the process? Start by providing
conditions for the first two to three days which are favorable to lactic acid
bacteria. A warm spot if you can easily manage one (but not too much higher
than 80ºF), and a reasonably high hydration (at least 100%). Use pineapple
juice if you like, to bypass the first round of bacteria. Feed with whole grain
flour until yeast are actively growing, not for the wider spectrum of sugars it
may offer, but for its higher numbers of yeast and lactic acid bacteria to seed
each phase in its turn. Don't feed too much or too frequently, so as to allow
the acids to accumulate and the pH to fall more rapidly. The ideal feeding
quantity and frequency would depend on the temperature, hydration, and how fast
the pH is falling. However, I usually recommend once a day at room temperature,
simply because it is the easiest to manage, it works, and the daily
manipulation helps to keep mold from getting started. Mold is the biggest
stumbling block for procedures in which a young mixture is allowed to sit idle
for two or three days at a time. Turning surface mold spores into the center by
re-kneading or stirring and scraping down the sides daily, is the best way to
get around it. Mold is not inhibited by low pH or pineapple juice, and
anti-mold properties don't fully develop until sourdough is well established.

While you don't actually need a formula to do this, no article on making
sourdough starter would be complete without one. This procedure was designed
with simplicity in mind, to be efficient and minimize waste. It was developed
with the participation of four willing and very patient women whom I worked
with online---DJ Anderson, Karen Rolfe, Deanna Schneider and the
still-anonymous 'lorian,' whose plea for help is what renewed the quest to find
a better way. I learned a great deal from the feedback they gave me as we
worked out the kinks, and this formula is a tribute to them.

There is nothing magic about the two tablespoons of measure used throughout the
first three days. Equal weights didn't provide a high enough ratio of acid to
flour to suit me, and equal volumes did. Two tablespoons is enough to mix
easily without being overly wasteful (and just happens to be the volume of an
eighth-cup coffee scoop, which is what I kept on the counter next to the flour
and seed culture for quick, easy feeding). These first few days don't really
benefit from being particularly fussy with odd or precise measuring, so make it
easy on yourself. Keep it simple, and let Mother Nature do the rest.

Day 1: mix...
2 tablespoons whole grain flour* (wheat or rye)
2 tablespoons pineapple juice, orange juice, or apple cider

Day 2: add...
2 tablespoons whole grain flour*
2 tablespoons juice or cider

Day 3: add...
2 tablespoons whole grain flour*
2 tablespoons juice or cider

Day 4: (and once daily until it starts to expand and smell yeasty), mix . . .
2 oz. of the starter (1/4 cup after stirring down-discard the rest)
1 oz. flour** (scant 1/4 cup)
1 oz. water (2 tablespoons)

* Organic is not a requirement, nor does it need to be freshly ground.
** You can feed the starter/seed culture whatever you would like at this point.
White flour, either bread or a strong unbleached all-purpose like King Arthur
or a Canadian brand will turn it into a general-purpose white sourdough
starter. Feed it rye flour if you want a rye sour, or whole wheat, if you want
to make 100% whole wheat breads. If you're new to sourdough, a white starter is
probably the best place to start.

On average, yeast begin to grow on day 3 or 4 in the warmer months, and on day 4
or 5 during colder times of the year, but results vary by circumstance. Feed
once a day, taking care not to leave mold-promoting residue clinging to the
sides or lid of your bowl or container, and refer back to the different phases
to track progress. Once you have yeast growing (but not before), you can and
should gradually step up the feeding to two or three times a day, and/or give
it bigger refreshments. This is the point at which I generally defer to the
sourdough experts. There are several good books on sourdough which address the
topic of starter maintenance and how to use it in bread. Just keep in mind that
the first days of the seed culture process have nothing to do with developing
flavor or even fostering the most desirable species. The object is simply to
move through the succession and get the starter up and running. The fine-tuning
begins there. Once yeast are growing well, choose the hydration, temperature
and feeding routine that suits you, and the populations will shift in response
to the flour and conditions that you set up for maintenance.

One more thing I have found is that with regular feeding at room temperature,
new starters seem to improve and get more fragrant right around the two week
mark. Maybe this coincides with the appearance of Lactobacillus
sanfranciscensis mentioned previously. It is generally regarded as the most
desirable species, as well as the one found to be the most common in
traditional sourdough.[7] A Fifth Phase? Obviously, there is still more to
learn.

References
1. Choi, In-Kwon, Seok-Ho Jung, Bong-Joon Kim, Sae-Young Park, Jeongho Kim, and
Hong-Ui Han. 2003. Novel Leuconostoc citreum starter culture system for the
fermentation of kimchi, a fermented cabbage product. Antonie van Leeuwenhoek
84:247-253.
2. Kurose, N., T. Asano, S. Kawakita, and S. Tarumi. 2004. Isolation and
characterization of psychotrophic Leuconostoc citreum isolated from rice koji.
Seibutsu-kogaku Kaishi 82:183-190.
3. Doyle, Michael P., Larry R. Beuchat, and Thomas J. Montville. 2001. Fruits,
Vegetables, and Grains, p. 135. Food Microbiology Fundamentals and Frontiers,
2nd ed. American Society for Microbiology Press, Washington, DC.
4. Katina, Kati. 2005. Sourdough: a tool for the improved flavour, texture and
shelf-life of wheat bread, p. 23.VTT Technical Research Centre of Finland.
5. Wing, Daniel, and Alan Scott. 1999. Baker's Resource: Sourdough Microbiology,
p. 231. The bread Builders. Chelsea Green Publishing Company, White River
Junction, VT.
6. Doyle, Michael P., Larry R. Beuchat, and Thomas J. Montville. 2001. Spores
and Their Significance, p. 50. Food Microbiology Fundamentals and Frontiers,
2nd ed. American Society for Microbiology Press, Washington, DC.
7. Arendt, Elke K., Liam A.M. Ryan, and Fabio Dal Bello. 2007. Impact of
sourdough on the texture of bread. Food Microbiology 24:165-174.