Sunday, March 27, 2022

Yeast - the Ubiquitous, Universal Fermenter

 Yeast - the Ubiquitous, Universal Fermenter  

Dr Abe V Rotor

Basi wine making, first day of fermentation. Note evolving gas - carbon dioxide. Wine is produced by anaerobic fermentation.

Bottled basi wine for tourists shops and for export. Basi was among the items carried between the Philippines and Europe via Acapulco, Mexico, during the Galleon Trade era (17th and 18th century) when the Philippines was a colony of Spain, so with Mexico. Old folks attribute the unique fine taste of basi to bubud (homemade yeast).

All kinds of alcoholic drink contain ethanol or ethyl alcohol - the only edible alcohol. Wine is as old as civilization. Serendipity must have led to early wine making techniques, the key being the domestication of  the first microorganism - Saccharomyces, the ubiquitous yeast.

Wine making is converting sugar into ethanol. Ethanol or ethyl alcohol (C2H5OH) is universal in all alcoholic drinks from beer to table wine to liquor, irrespective of generic or brand name. The strength of wine is indicated by proof, which is actually twice that of its percentage content.

Thus, 80 proof is 40 percent ethanol, which is the strength of Vodka, some Brandy, Cognac, Whiskey, and the like. The strength of beer normally ranges from 3 to 5 percent, unless fortified with distilled ethanol to raise it to say, 7 percent. Fortified wine is also common.

Natural table wine such as Basi of the Ilocos region, table wine from grapes and other fruits, normally contain 10 to 12 percent ethanol. Beyond this level, the fermenting yeasts simply die off from the accumulation of ethanol - a biological phenomenon called autotoxicity. It means that the yeasts are killed by their own product, often leaving behind the unfermented sugar. It is this residual sugar that makes a wine sweet - naturally, that is.

Ripe leaves of samat or binuga (Cananga tenarius) is used in making basi. 

Yeast is found in ripening leaves. Here the leaves contain the highest level of sugar which the plant did not use or store. The yeast acts of the sugar and as the leaves fall to the ground, a myriad of microorganisms and animals (from fungi to earthworms and grazing animals) obtain their energy from them. Ultimately the organic matter left behind becomes part of the soil, releases the needed nutrients to the growing plants and those in the next generation.

Yeast is ubiquitous, it is found in flowers, ripening fruits, honeydew, ripening leaves. It comes in different species under the genus Saccharomyces, among them cerevisiae and ellipsoides. There are also other genera such as Brettanomyces and Debaryomyces. Not all yeasts make good wine. But one thing is universal to them. It is Nature's way of converting sugar molecules (C6H12O6) back to their elemental form.  Oxidation often accompanies such process, thus converting ethanol to acetic acid (CH3COOH), which is vinegar. Vinegar actually is a term, vin-egar, which means sour wine.

Natural vinegar is oxidized ethanol, usually with the aid of bacteria, principally Acetobacter and Leuconostoc. The latter forms gelatinous capsule that accumulates into a transparent to white layer we call nata. This is the principle involved in making Nata de coco and nata de piƱa.

So, even before sugar ferments to vinegar, nata bacteria and other contaminants can spoil wine and vinegar making. A host of organisms are soon attracted such as Drosophila flies, blue bottle flies, wasps, moths and butterflies that feed on the spoiled must. This is happening to unharvested fruits in the field, to remnants of pollination and fertilization, It is true in ponds and lakes where biomass of algae die of algal bloom. Ultimately the product is simply water, evaporating into the air or settling down into a pool or seeping into the ground, and all the organic compounds once part of the living world are back to their elemental components ready to be reassembled into the next living generation. Indeed this a great wonder on how Nature keeps a dynamic balance of the environment called homeostasis.

We can only imagine the ingenuity of wine makers far back during the Epyptian civilization, and in the Orient, the Chinese civilization. I had a chance to visit the ruins of an Assyrian fort outside Tel-Aviv. There, our guide pointing at broken tall jars, said, "The Assyrians were among the best wine makers in the ancient world." The Assyrians were powerful, not even Ramses could conquer them, They had a flourishing economy. Their vineyards can be glimpsed from the vineyards around the place Lakish today. Lakish wine is well-known all over the world, perhaps as famous as the Bordeaux in France.

Without yeast, our world would be a less happy one. Perhaps many organisms wouldn't be around in the first place, including us humans.

Here's a toast to the wonderful yeast. Cheers! KampaiMabuhay!~

Part 2  Basi: Preparation of Bubod – Yeast Complex

http://                                Closeup of bubud, homemade yeast complex.

Here is a list of yeast isolates from plants growing at the SPCQ garden. The author developed the combined process of isolation, multiplication, identification and banking.

Researcher/Plant Source/Wild Yeast

1.Muega, N Basil (
Ocimun basilicum), Debaryomyces,
Indonesian Queen - Justicia (Trichosporon genderossa), 
and kamuning Kloerckera
(Murraya pinnaculata)

2.Valdez, M.M Guava (Psidium guajava) Brettanomyces
Powderpuff (Calliandra cergenila) Debaryomyces
Aratilis (Muntigia calabura) Saccharomyces ,
Trichosporon


3.Lacap, DC Duhat (S. cumini) Saccharomyces
Saccharomycodes, Debaryomyces,
Hansenula
Kloeckera
Nematospora

4. Ngo, LM Coconut (Cocos nucifera) Cryptoccocus, 
Hansenula

The isolation of these wild yeasts was made possible using a technique developed in the laboratory of SPUQC. Food Development Center of NFA analyzed and identified the yeast isolates.


The Protocol named after the author is summarized in three parts, as as follows:

Part 1 - Isolation and Identification of Wild Yeasts from Plants

1. Determination of possible sources of wild yeasts. Since yeast is ubiquitous, it is likely found in places where there is a ready supply of sugar. Pollen and nectarines of flowers offer such as an ideal place.

2. Yeast cells are isolated from these floral parts and inoculated in a 15 percent sucrose solution contained in sterilized bottles (3/4 volume). Beer bottles are preferred because their brown or green color protects the isolates from UV radiation.

3. The bottles are plugged with sterilized cotton and are kept in a dark, cool chamber for at least three days.

4. As fermentation takes place, carbon dioxide evolves and in the process creates a CO2-rich chamber in the bottle that serves as an aseptic blanket especially against aerobic bacteria.

5. The culture is then analyzed in the laboratory. Identification of the yeast isolates is done using the standard procedure of FDC. Yeast isolates by Muega et al at SPUQ were obtained using this procedure.

6. The next step is the isolation and culture of the desired yeast isolates for specific purposes. However, the yeast complex as a whole, after proper identification, can be propagated for commercial use.


Part 2 – Propagation and Banking/Storage of the Yeast Isolates

1. The yeast complex is allowed to multiply for another 5 days in the culture bottles. Detection of any contaminant necessitates the discarding the culture, and the procedure is repeated.

2. Rice flour is heated to 100 degrees Celsius and allowed to cool to 50 to 60 degrees Celsius (equivalent to pasteurization). The fermenting sugar solution is the mixed with the flour to make dough. Ground dry ginger is added at the rate of 1 part to 4 parts rice flour. The dough is mashed thoroughly and made into balls, two inches in diameter.

3. The balls are laid on cheesecloth, lined with clean rice straw, and incubated in a wooden box for 5 to 6 days in a dark, enclosed chamber, at 35 to 40 degree Celsius.

4. The balls are air-dried for 3 to 5 days, or until they are dry enough to be pulverized. Direct sunlight may kill the yeast cells. This is now the inoculant that is used in basi fermentation. The rate of inoculation is equivalent to 10 balls to a standard size jar.

5. The powdered inoculant can be stored in an airtight glass container and placed in the vegetable section of a refrigerator. Viable storage time is around 6 months.


Part 3 – Alternative Procedures and Other Applications of the Protocol

1. Substrates may vary, according to the microorganisms to be propagated and banked. Papaya pulp is commonly used for Aspergillus niger as shown in the experiment of Marasigan, 1995.

2. Papaya pulp is also recommended in the preparation of Rhizobium inoculant for soybeans and other legumes (Jacob 1997)

3. Other fruit pulps such as citrus and mango have been tried successfully in the propagation of food-fermenting organisms such as Leuconostoc mesenteroides (nata de coco), Lactobacillus (yogurt), Micrococcus and Pediococcus (patis), and other Halobacteria (bacteria responsible in bagoong making). (8)

4. Other alternatives the protocol can adopt are in the propagation of
cellulose-breaking bacteria such as Trichoderma, and biological pesticide such as Bacillus thuringiensis. 

Part 3 - A visit to an 18th century Basi wine cellar 
San Vicente, Ilocos Sur 
 
Dr Abe V Rotor

Original basi wine cellar and jars (burnay) date back to the
 18th century across six generations of continuous operation, 
interrupted only by the Second World War for five years. 
 
The cellar attracts researchers, students and tourists for its historical 
significance with the Spanish Galleon Trade, and technology o
the old folks in making basi and its related products, principally 
vinegar (sukang Iloko).  

By now this jar of basi is 13 years old.  Unless opened, it remains longer 
in aging. The general rule is, the longer wine is aged, the more mellow it 
becomes.  It's not really so.  There are other factors to consider like 
damaged clay cap and leaching.  And there's the basic rule that "only 
good wine mellows with age" (So with man, they add.) 

Crystalline golden color and pleasant wine aroma meet the happy
connoisseur after the desired aging period is reached (at least two 
years in the case of Rotor basi). Fresh and direct from the jar, the 
harvest is bottled, sealed and labelled (as shown below), according
 to customers' specifications intended for an occasion like
 wedding, Christmas, exhibit, and the like. 


House guest picks a fruit wine of his choice,  Fruit wine making 
follows the basic fermentation-aging process in basi making.
There are 20 kinds of fruit wine developed in this cellar from different 
fruits growing locally like macopa, aratiles, duhat and guyabano. 

Stamp commemorating the Galleon Trade. 
Scene of a Galleon trading post in Ciudad de Vigan in Spanish time. 

"The Manila-Acapulco galleon trade* in the 18th century was undertaken by Vigan Chinese mestizo traders who exported local products such as basi, tobacco and abel to Europe and other parts of Asia..." Pia Roces Morato, Thorns and Roses


                                      * Manila-Acapulco galleon trade 1565–1815

                                
Basi jar lying on the sea floor where a galleon ship was wrecked. 

Basi sparked one of the major revolts against Spanish rule by the natives when wine monopoly was declared by the government. This meant virtually taking the industry from the hands of the natives. The short-lived uprising took place in Ilocos, with the final battle fought on both sides of the Bantaoay River which runs through the towns of San Vicente up to San Ildefonso, which are today the major suppliers of Basi principally to tourists in Vigan, UNESCO's world heritage city, and one of the cultural wonders of the world.
                             

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