Rosita. 2008.

Produksi etanol dari onggok menggunakan ekstrak kasar enzim alfa amilase,
glukoamilase dengan Saccharomyces cerevisiae. [Tesis]. Bogor [ID]. Program Studi Magister
Bioteknologi Sekolah Ilmu Teknologi Hayati. Institut Pertanian Bogor

Hui Y.H. 2006. Bakery Products: Science and Technology 1st Edition. Blackwell Publishing : Iowa-
USA.

Starter cultures may be defined as concentrates of living microorganisms that are used to accelerate
and standardize the preparation of fermented foods. Baker's yeast is the main microbial starter for
the preparation of bakery products. Besides dough fermentation and bakery applications,
commercial yeast is used for the production of beer, alcohol, wine, and various fermented dairy
products. Saccharomyces cerevisiae is the main baker’s yeast species for bread fermentation.

Yeast or leaven is still the term used to describe the starter of a series of events leading to dough
leavening and bread. However, scientific knowledge teaches us that amylases first degrade wheat
starch into smaller units such as glucose and maltose, which is then converted by yeast cells into gas
and alcohol through yeast enzymes (meaning within yeast).

Inside fermenting dough or food, anaerobic conditions rapidly prevail because oxygen is consumed
by living microorganisms (yeasts and others). Fermentation has been described by Pasteur as “la vie
sans air” (life without air) so fermented foods have generally been described as foods processed
through anaerobiosis.

Commercial baker’s yeast is made from one specific yeast species, Saccharomyces cerevisiae, which
is part of a group of yeasts called Ascomycetes. According to Kurtzman and Fell (1998), there exist
about 100 genera and more than 700 species of yeasts. Fourteen yeast species currently belong to
Saccharomyces (= sugar mushroom), including species cerevisiae (= beer). Around the end of the
19th century, these names were given to describe this special microorganism that was isolated from
beer and grew rapidly in sugar solution. Members of this yeast species easily survive under aerobic
or anaerobic conditions, which is not the case for all yeasts. When oxygen is available, S. cerevisiae
multiplies very well; without oxygen, it rapidly excretes gas (mainly carbon dioxide) and alcohol
(mainly ethanol).

According to Gelinas and Houde (1998), baker’s yeast preparations are mainly used for gas
production in bakery applications, and ethanol and aroma production in alcoholic fermentations
(beer, alcohol, wine).

Around 1900, just before the advent of commercial baking, household recipes for bread
fermentation starter or leaven (often called “yeast”) were often based on natural fermentation of
sugar compounds of food such as fruits.

Baker’s yeast (Saccharomyces cerevisiae) can grow on glucose, fructose, and sucrose. It easily
transforms sucrose into glucose and fructose but generally needs some adaptation period before
assimilating maltose. Yeasts cannot assimilate lactose (a disaccharide present in milk, formed from
glucose and galactose) or raffinose (a trisaccharide present in molasses, formed from galactose,
glucose, and fructose) (Vaughan-Martini and Martini 1998)

Excess of sugar (more than 0.01%) leads to a shift of the yeast metabolism called the Crabtree effect
(Polakis and Bartley 1965): if baker's yeast is grown with excessive amounts of glucose, its oxidative
activity will rapidly be inhibited, and lower biomass yields will be obtained because ethanol and gas
will be formed instead of cell biomass. Nitrogen and phosphorus must also be thoroughly controlled
because their concentration may vary with molasses source (Harrison 1971). As reviewed by Chen
and Chiger (1985), optimal yeast cell yield would be highest at 28.5-32.5"C and pH 4.1 (Reed and
Nagodawithanam 1991).

Yeast is grown in liquid media containing about 0.7% yeast solids at the beginning of the last
production step. At the completion of the fermentation, yeast solids concentration has increased by
a factor of about 8 to give about 56% yeast solids in the fermentation medium or 10-13% by cell
volume (Reed and Nagodawithana 1991).

Yeast activity is very low at 4"C, and cells are killed around 45-50°C. During dough fermentation,
yeasts and lactic acid bacteria form gas and acids that will slightly lower pH, considering the high
buffering capacity of dough constituents. Such an increase in acidity will slow down yeast
fermentation, but this will not be critical except for very long fermentation periods.

The contribution of baker’s yeast to aroma production in dough fermented for about 2.5 hours at
27°C with 2.5% yeast (flour basis) has been determined in dough (Frasse et al. 1993) and bread
(Frasse et al. 1992). According to Zehentbauer and Grosch (1998), high yeast concentration in dough
would enhance the concentration of “roasty” smelling, while fermentation at low temperature (4°C
for 18 hours) favored the production of a malty-type aroma.

Examples of Baker’s Yeast Strains for Specific Dough Applications (Flavor)

Bread aroma enhanced using wine yeast (McKinnon et al. 1996)

Reference for Part 9. Yeast by Gelinas P. (Part II. Major Baking Ingredients)

Chen SL, Chiger M. 1985. Production of baker’s yeast. In: Blanch HW, Drew S, Wang DIC, editors.
Comprehensive Biotechnology, vol. 3. Oxford: Pergamon Press. Pp. 429-461.

Frasse P, Lambert S, Levesque C, Melcion D, RichardMolard D, Chiron H. 1992. The infience of

fermentation on volatile compounds in French bread crumb. Lebensm -Wiss u -Techno1 25:66-70.

Frasse P, Lambert S, Richard-Molard D, Chiron H. 1993. The infience of fermentation on volatile

compounds in French bread dough. Lebensm Wiss u Technol 26:126-132.

Gelinas P, Houde A. 1998. Catalog of Food Microbial Starters in North America. Saint-Hyacinthe, QC:
Food Research and Development Centre, Agriculture and Agri-Food Canada.

Gelinas P, Lagimonikre M, Dubord C. 1993. Baker’s yeast sampling and frozen dough stability. Cereal
Chem 70:219-225.

Harrison JS. 1971. Yeasts in baking: Factors affecting

changes in behaviour. J Appl Bacteriol 34:173-179.

Kurtzman CP, Fell JW. 1998. The Yeasts, a Taxonomic Study, 4th ed. Elsevier: Amsterdam.

McKinnon CM, Gelinas P, Simard RE. 1996. Wine yeast preferment for enhancing bread aroma and
flavor. Cereal Chem 73:45-50.

Polakis ES, Bartley W. 1965. Changes in the enzyme activities of Saccharomyces cerevisiae during
aerobic growth on different carbon sources. Biochem J 97284-297.

Reed G, Nagodawithana TW. 1991. Yeast Technology, 2nd ed. New York: AVI.

Vaughan-Martini A, Martini A. 1998. Saccharomyces Meyen ex Reess. In: Kurtzman CP, Fell JW,
editors. The Yeasts, a Taxonomic Study, 4th ed. Elsevier: Amsterdam. Pp. 358-371.

Zehentbauer G, Grosch W. 1998. Crust aroma of baguettes. 11. Dependence of the concentrations of
key odorants on yeast level and dough processing. J Cereal Sci 28:93-96.

Major Quality Control Criteria for Baker's Yeast

Composition : No pathogens, Low Bacteria and wild yeasts counts, low quantity of reducing
compounds (glutathione)

Appearance : Light Color

Activity : High Gassing power, Good keeping properties, Maltose assimilation, Acceptable flavor (in
bread), Tolerance to acidity-antimicrobials-salt-freezing-or osmotic pressure (sweet dough), Active
at low or high dough temperature

Ease of Use : Good dispersion in water (rapid rehydration), may be pumped (liquid)

Economy : Low Cost

XLSTAT versi 2021.2.1.1127

Table.1 Outline of Major Food Fermentations involving Yeast

Not only the water in beverages, but all waters used in food production, washing, processing,
extracting, liquid starter fermentation and baking are called drinking waters. The Handbook of
Drinking Water Quality (De Zuane 1997) sets guidelines for waters used in baker’s yeast.

De Zuane J. 1997. Handbook of Drinking Water Quality, 2nd ed. Van Nostrand Reinhold

SUGAR
Sugar is the major source of energy for the yeast in fermentation. Appropriate amounts of sugar
help significantly in the yeast fermentation process. In the general recipe, a sugar content of 5% will
increase yeast fermentability and enhance fermentation. However, when the sugar content is over
8% in the recipe, the osmotic pressure produced destroys the yeast cells and will slow the speed of
fermentation significantly. For example, the sugar content in recipes for sweet baked goods in
Taiwan can be as high as 20-25%. Because of the dramatic increase in the osmotic pressure, it
significantly retards yeast fermentation, and the time for fermentation must be extended. In
general, in the making of sweet baked goods, yeast specifically produced for use with high sugar
content can be used and the quantity of yeast increased, thus shortening the fermentation time.

YEAST There are three kinds of yeasts commonly used in breadmaking: compressed yeast, active dry
yeast, and instant active dry yeast. In terms of fermentation speed, the order is: compressed yeast >
instant active dry yeast > active dry yeast. The active dry yeast is the strongest in terms of
fermentability and is more capable of tolerating osmotic pressure and the propionate preservatives
used in baked goods; the instant active dry yeast is the strongest in terms of activity of the yeast. In
terms of the fermentation environment for the yeast, the appropriate pH is between 4 and 6, and
the appropriate temperature is between 35 and 38°C (95 and 100.4"F). In this environment, the
yeast is able to produce the greatest amount of gas. During active yeast fermentation, abundant
alcohol is produced. This alcohol is able to dissolve the yeast cells and thus slow fermentation speed.
Also, the dead dry yeast cells contain glutathione, which will weaken the gas retention capacity of
the gluten.

Yeast is a single cell fungus with about 1.5'' cells/g. There are many different types of yeast, but the
one used for fermentation of dough is Saccharomyces cerevisiae. Under anaerobic conditions, with
the exclusion of oxygen, this organism is capable of the production of carbon dioxide gas and alcohol
from lower sugars. It is its gas production ability that is of most importance in the fermentation of
dough.

Yeast is a living organism, it is sensitive to temperatures (Fig. 1.3 Effect of temperature on yeast).
Yeast is active between 0 and 55°C. At temperatures less than 20°C and over 40 °C, the rate of
growth is significantly reduced. Yeast can survive for a few weeks at temperatures as low as -20°C,
but it gradually loses its fermentation capacity. The most favorable temperature for yeast to multiply
is between 20 and 27°C; optimum multiplication of yeast is achieved at around 26°C. The optimum
temperature for fermentation lies between 27 and 38°C; yeast ferments best at 35°C (Schunemann
and Treu 1984)

Yeast is a living organism that will act in conditions with or without oxygen. In the bread recipe with
sugar added, the energy source provided to the yeast for fermentation comes from two sources: (1)
added sugar in the bread recipe, which provides available carbohydrates to the yeast for the first
stage of the fermentation; and (2) maltose, from the hydrolysis of damaged starch by amylases.

In the disjunction of sugars by the fermentation process, yeast converts available carbohydrates to
large quantity of carbon dioxide gas until reaches the point of saturation. The decrease of pH
enables the yeast, improvers, and enzymes in the dough to become more active.

NATURAL SPONGE

In recent years, under the tide of natural food, the call for healthy natural sponge is receiving more
attention, and in Japan, many bakeries specializing in selling breads made from natural sponge have
emerged. The natural sponge uses the natural yeast that adheres to fruits, flour, and rye flour to
make a sponge. Besides the yeast that naturally adheres to the fruits, flour, and rye flour, the natural
sponge also contains lactic acid bacteria and acetic acid bacteria (which cause natural sponge to be
referred to as compounded sponge). These bacteria produce organic acids during the fermentation
process and provide the breads with rich flavor, sour taste, and other qualities, revealing the special
characteristic of the natural sponge.

Because the fermentability of the natural sponge is comparatively weak, it is necessary to add
sucrose to help cultivate the natural sponge, increase its strength, and increase its fermentation
activity. Such complicated procedures are more suitable for adoption by the small bakeries or
specialized shops; the use of natural sponge is not suitable for mass production. However, breads
produced by using natural sponge are not only healthy, but also possesses special character,
individualized flavor and odor. These are the driving forces attracting people to become more
actively involved in developing natural sponge.

The natural yeast used for breadmaking comes mainly from fruits and grains. Recently, the most
commonly known ones are the fruit sponge, hop sponge, sour sponge, and alcohol sponge. Using
natural yeast in breadmaking has the problem of lack of control over the fermentation. This is
because the added natural sponge changes according to its own condition, temperature, and
humidity, which leads to different results in the fermenting condition.

SUGAR

Sugar is the major source of energy for the yeast in fermentation. Appropriate amounts of sugar
help significantly in the yeast fermentation process. In the general recipe, a sugar content of 5% will
increase yeast fermentability and enhance fermentation. However, when the sugar content is over
8% in the recipe, the osmotic pressure produced destroys the yeast cells and will slow the speed of
fermentation significantly. For example, the sugar content in recipes for sweet baked goods in
Taiwan can be as high as 20-25%. Because of the dramatic increase in the osmotic pressure, it
significantly retards yeast fermentation, and the time for fermentation must be extended. In
general, in the making of sweet baked goods, yeast specifically produced for use with high sugar
content can be used and the quantity of yeast increased, thus shortening the fermentation time.

Bastetti G. 2001. Technical Bulletin. American Institute of Baking, vol. 23. Manhattan, KS: American
Institute of Baking.

The reaction formula is as follows:

C6H12O6 + 2CO2 + 2C2H5OH + 27 kcal

Alexander RJ. 1998. Sweeteners: Nutritive. St. Paul, MN: Eagan Press.

Doerry WT. 1995. Breadmaking Technology. Manhattan, KS: American Institute of Baking. Chapters
4, 5, and 6, pp. 62-162.

Nelson AL. 2000. Sweeteners: Alternative. St. Paul, MN: Eagan Press.

Schunemann C, Treu G. 1984. The leavening of wheat-flour doughs. In: Shewchuk R, editor. Baking:
The art and science. Calgary, Alberta, Canada: Baker Tech Inc. Pp. 37-47.

Sourdough
Before commercially prepared yeast was widely available, breadmaking was often started by mixing
flour and water and letting this mixture stand until wild airborne yeasts settled on it and began to
ferment it. This “starter” was then used to leaven bread. A portion of the starter was saved, mixed
with more flour and water, and set aside to leaven the next batch of bread. This process is still used
today, although the sours are generally started with commercial yeast.

The sour is actually a kind of sponge, except that it is allowed to ferment until it becomes strongly
acidic. It may be used in two ways: (1) as a sponge in the production of the bread dough (except that
a portion is saved for the next bread production) or (2) as an addition to a straight dough as flavoring
(e.g., light sour or rye sour).

Microorganisms in sourdoughs consist of two different types, bacteria and yeast. The end products
of bacterial fermentation are primarily organic acids, and yeasts produce mainly alcohol and carbon
dioxide. The flavor comes from the various organic acids produced by bacteria, which may or may
not react with each other or with the alcohol from yeast fermentation to form flavorful esters during
the baking process. The most desirable organic acids are produced by heterofermentative lactic acid
bacteria. The kind of acid produced depends on the type and temperature of the material
fermented. Generally, lower fermentation temperatures favor the formation of desirable organic
acids, such as acetic, citric, lactic, fumaric, malic, and others. Higher fermentation temperatures
seem to favor the formation of butyric and slightly longer chain fatty acids, which tend to result in
undesirable flavors. The famous San Francisco sourdough contained a specific lactic acid bacterium
(L. sanfrancisco) and a unique yeast strain that thrives under high acid (low pH) conditions and is
unable to ferment maltose.

“Sourdough”, a mixture of flour and water fermented with lactic acid bacteria (LAB) and yeast, is the
basis for sourdough bread production. Sourdough bread has a long tradition in Europe. Today it is
still popular among big rye producers like the Russian Federation, Poland, Germany, Belarus,
Ukraine, Lithuania, Denmark, and others.

In recent years there has been a growing interest in sourdough bread production based on the
motto “good health with good taste.” The importance of sourdough bread is demonstrated by the
existence of specific legislation regulating its production in Germany and in France (Decret no. 97-
917, 1997; Seibel 2001).

It is not known where and when sourdough was used for the first time. It is believed that ancient
Egypt was the birthplace of this invention. In the literature a predominant hypothesis is that
sourdough bread can be dated from the 26th century BC (Schickentanz and Davidson 1996).
However, sourdough bread became popular only after the Hebrews left Egypt (Spicher 1983, Lonner
1988).

The art of sourdough preparation spread throughout the Mediterranean region by the end of the
18th century BC. After Egyptians and Jews, the Greeks, who treated sourdough bread as a privilege
and consumed it only on feasts, adopted this skill (Schickentanz and Davidson 1996).

Sourdough was commonly used as a leavening agent until baker’s yeast was introduced in the
middle of the 19th century (Spicher 1983, Lonner 1988). Baker’s yeast simplified the leavening
process and allowed automation in industrialized bakeries. The trend toward reduced use of
traditional fermentation techniques in baking applications was reversed during the past decade
(Ganzle and De Vuyst 2004).

Saccharomyces cerevisiae, characteristic mainly for rye sourdoughs, is a top fermenting yeast with
an optimum growth temperature of 26-32°C. It is an aerobe or facultative anaerobe. It utilizes
glucose, maltose, galactose, sucrose, and partly raffinose (Spicher and Stephan 1993, Gobbetti
1998).

In the course of fermentation, yeast plays several important roles. First, it produces carbon dioxide,
which expands the dough, resulting in the proper porosity of the crumb and the proper volume of
bread. Besides, yeast produces a lot of side products such as aldehydes, aliphatic and isoaliphatic
alcohols, acids, keto acids, and esters, which alone or in combination with other compounds can
create specific and unique fivors of bread (Hansen and Hansen 1994, Makoto et al. 1990).

BAKERY STARTER CULTURES

In fermented food, both the quality of the microkra and metabolic activity depend on the
composition of the raw material, water activity, redox potential, pH, temperature, and the presence
of antagonistic microorganisms and bacteriophages. Thus, the type of raw material and the applied
technology determine the final product of fermentation. In traditional fermentation processes,
microorganisms undergo changes after which only a few strains acquire dominance and control of
the process. Succession is relatively repeatable, but may be in fienced by bacteriophages, inhibitors,
or lack of the desirable microorganisms in the raw material (Hammes and Tichaczek 1994, Foschino
et al. 1995).

Possible variations in the development of microflora in spontaneously fermenting sourdoughs do not

guarantee the desired quality in the final product. Thus, spontaneous fermentation is often
optimized through backslopping, that is, inoculation of the raw material with a small quantity of
sourdough from a previously performed successful fermentation (Leroy and De Vuyst 2004). This
procedure of adding a portion of mature full sour at the beginning of fresh sourdough preparation is,
however, the unconscious form of using selected strains of lactic acid bacteria and yeast. Although
backslopping is still in use, the large-scale production requires more reliable methods to control the
sourdough fermentation process and reduce the risk of fermentation failure, for example, bakery
starter cultures (Diekmann and Seiffert 1992).

The use of such a starter guarantees (Cossignani etal. 1996):

a. Initiation of the fermentation process,

b. Obtaining the desired baking parameters of the dough due to the biochemical changes in
fermented flour,

c. Stability of the fermentation process,

d. Shortening and simplifying sourdough production,

e. Obtaining the unique and very attractive taste and fivor of the bread,

f. Bread of high nutritional value,

g. Reduction or elimination of artificial improvers and preservatives,

h. Repeatability of bread quality, and

i. Prolonged shelf life of sourdough bread.

Bakery starter cultures are defined as selected lactic acid bacteria in the form of a pure culture or a
mixed culture, with yeast (Bode and Seibel 1982). To some extent, backslopping was the first form of
bakery starter culture (Spicher 1995, Leroy and De Vuyst 2004). However, modern starter cultures
consist of carefully selected strains with well-defined metabolic characteristics, which allow the
design of mature sourdough and, consequently, development of sourdough bread of the desired
quality. Typical starter culture consists of 10^7-10^11 CFU/g lactic acid bacteria and 10^5-410^6
CFU/g yeast (Yondem et al. 1992, Hochstrasser et al. 1993)

Traditional sourdough bread technology is based on a spontaneous fermentation process from lactic
acid bacteria and yeast occurring naturally in kur. The growth of microorganisms occurs in several
stages and lasts for up to 10 hours, depending on the temperature. The character of the process
results from growth of microorganisms in different environmental conditions (Barber et al. 1991,
Yondem et al. 1992)

HEALTH BENEFIT

Health benefits of sourdough bread may be reviewed in many aspects. Bread attractiveness
determines the decision to purchase but also initiates numerous physiological processes to promote
digestion. Sourdough bread digestibility is enhanced by enzymatic processes that take place during
fermentation and increase the content of easily available carbohydrates and proteins.

However, after consumption of sourdough bread, satiety is achieved faster, which can be explained
by a lower rate of stomach emptying and slower starch digestion. This effect is related to the
presence of lactic acid during baking, which alters starch-gluten interactions. It strongly affects
postprandial blood glucose and insulin responses, lowering glycemic index (GI) of sourdough bread
(Ostman et al. 2002a,b). Thus sourdough bread meets recommendations for consumption of low GI
food (FAO-WHO 1998), suggesting a protective role against the development of non-insulin-
dependent diabetes mellitus and cardiovascular disease (Bjorck et al. 2000). The therapeutic effect
of such foods is extremely important, especially in the case of patients with already diagnosed
diabetes (Salmeron et al. 1997, Jarvi et al. 1999). Health benefits of LAB development in sourdough
are based mainly on lactic acid production, which results in a decrease in pH below the point at
which undesirable bacteria can grow. In the human digestive tract this factor controls the quality of
intestinal microkra, ensuring the proper course of digestion and excretion (Fleming et al. 1985)

Anaerobic conditions and a low pH preserve bioactive labile compounds, such as vitamins, plant
sterols, or p-glucan, present in the raw material. Some strains even raise the content of B vitamins in
the fermented product. The nutritive value of rye flour fermented with lactic acid bacteria is
elevated due to an increase in ribofivin and niacin content, as well as exogenic amino acids, namely
lysin, tryptophane, and methionine (Lay and Fields 1981, Hammes and Tichaczek 1994).

The use of bakery starter cultures enables nutrient enrichment of bread. Lactic acid bacteria and
yeast convert minerals from the culture medium into bioavailable organic forms. Selenium
supplementation may serve as a good example of elevating the content of an element deficient in
the diet via sourdough bread enrichment (Diowksz et al. 1999, 2000).

There are also data indicating sourdough effectiveness in lowering serum triglyceride and cholesterol
levels as well as improving the ratio between HDL and LDL fractions. This effect may result from
exopolysaccharide production occurring during sourdough fermentation (Tieking et al. 2003). In
addition, acetic acid in sourdough bread can enhance the salty taste, which reduces the amounts of
salt used for bread production. This factor is of great importance, especially in the case of kidney
problems and hypertension.

Traditional sourdough fermentation is troublesome and economically ineffective. However,

negligence in the completion of the process results in serious bread failures, namely, leathery crust,
a typical flavor, crumbling, or dull taste.
Although both taste and aroma are finally created in an oven, these qualities are determined by the
sourdough fermentation process. At this stage of bread production, products of lactic acid bacteria
and yeast emerge, and they determine the unique sourdough flavor (Hansen and Hansen 1994,
1996; Gobbetti 1998; Seitz et al. 1998)

-------------------------------------------------------

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Bjorck I, Liljeberg H, Ostman E. 2000. Low glycaemic-index foods. Br J Nutr 83:S149.

Bode J, Seibel W. 1982. Sourdough and Guideline. Getr Mehl Brot 36:ll

Cossignani L, Gobbetti M, Damiani P, Corsetti A,

Simonetti MS, Manfredi G. 1996. The sourdough microfira. Microbiological, biochemical and
breadmaking characteristics of doughs fermented with freeze-dried mixed starters, freeze-dried
wheat sourdough and mixed fresh-cell starters. Z Lebensm Unters Forsch 203:88.

Decree no. 97-917. 1997. From Decree Concerning about what

concerns certain categories of bread the 1st of October, modifying the decree no. 93-1074 of 13
September 1993. France

Diekmann H, Seiffert M. 1992. Safe long-term sourdough production through the targeted use of
freeze-dried starter cultures. Getr Mehl Brot 3:76.

Diowksz A, Peczkowska B, Wlodarczyk M, Ambroziak

W. 2000. Bacteria-yeast and plant biomasses enriched in Se via bioconversion process as a source of
selenium supplementation in food. Food Biotechnology 17295.

FAO/WHO. 1998. Carbohydrates in human nutrition: Report of a joint FAO/WHO expert

consultation. FAO Food and Nutrition Paper 66:l.

Fleming HP, Mc Feeters RF, Daeschel MA. 1985. The

lactobacilli, pediococci and leuconostocs: Vegetable

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Cultures for Foods, p. 97. Boca Raton, FL: CRC

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Foschino R, Galli A, Perrone F, Ottogalli G. 1995.

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Ganzle M, De Vuyst L. 2004. Sourdough fermentation: From fundamentals to applications. Cereal

Foods World 49:42.

Gobbetti M. 1998. The sourdough microflora: Interactions of lactic acid bacteria and yeast. Trends
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Hammes WP, Tichaczek PS. 1994. The potential of lactic acid bacteria for the production of safe and
wholesome food. Z Lebensm Unters-Forsch 198:193.

Hansen A, Hansen B. 1994. Volatile compounds in wheat sourdoughs produced by lactic acid
bacteria and sourdough yeast. Z Lebensm Unters Forsch 198: 202.

Hansen A, Hansen B. 1996. Flavour of sourdough wheat bread crumb. Z Lebensm Unters Forsch 202:
244.

Hochstrasser RE, Ehret A, Geiges O, Schmidt-Lorentz

W. 1993. Microbiology of bread dough production. Chapter IV. Microbiological analysis of baking
agents and sourdough starter cultures. Mittel Gebeit Lebensm-Unters Hyg 84:622.

Jarvi AE, Karlstrom BE, Granfeldt YE, Bjorck IME,

Asp N-G., Vessby BOH. 1999. Improved glycemic control and lipid profile and normalized fibrinolytic
activity on a low-glycemic index diet in type 2 diabetic patients. Diabetes Care 22:lO.

Lay MM-G, Fields ML. 1981. Nutritive value of germinated corn and corn fermented after
germination. J Food Sci 46:1069.

Leroy F, De Vuyst L. 2004. Lactic acid bacteria as

functional starter cultures for the food fermentation

industry. Trends Food Sci Technol 15:67.

Lonner C. 1988. Starter cultures for rye sour doughs. Characteristics and functions of lactic bacteria.
PhD

dissertation. Lund University.

Makoto W, Kazuro F, Kozo A, Shigenori 0. 1990. Mutants of bakers’ yeasts producing a large amount
of isobutyl alcohol or isoamyl alcohol flavour components of bread. Appl Microbiol Biotechnol 34:
154.

Ostman E, Liljeberg Elmstahl HGM, Bjorck IME. 2002a. Barley bread containing lactic acid improves
glucose tolerance at a subsequent meal in healthy men and women. J Nutr 132:1173.

Ostman E, Nilsson M, Liljeberg Elmstahl HGM, Molin G, Bjorck IME. 2002b. On the effect of lactic acid
on blood glucose and insulin responses to cereal products: Mechanistic studies in healthy subjects
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HALAL-BAKING FOR MUSLIMS

One of the Shar’ah (divine laws) in Islam revolves around strict food laws. The holy book of Islam,
the Qur’an, forbids “the consumption of pork, blood, carrion, or food which has been sacrificed to
idols” (Van der Krogt 1990). In practice, Muslims will not eat products that have any traces of pig,
reptiles, carnivorous animals, or animals that are not slaughtered using the correct procedure.
Alcohol is also

forbidden. Foods that are suitable for consumption by Muslims are termed halal. Halal is defined as
“released from prohibition” and implies “that which is lawful” (Bowker 1998)

Van der Krogt C. 1990. Chapter 14. Islam. In: Donovan P, editor. Religions Of New Zealanders.
Palmerston North, New Zealand: Dunmore Press. Pp 186-205.

Bowker J. 1998. What Muslims Believe. Oxford; Washington, DC: One World Publishers. P. 176.

Consumer expectations for the variety and selection of foods available for purchase have risen as
per capita income has increased in both developed and developing countries. Customers are
prepared to pay more for organic foods and foods that not only provide nutrition but also contribute
to improved well-being. Products that are high in fiber and low in fat and sugar, and also contribute
to specific nutritional requirements need not be boring or a chore to eat. Healthy baked products
should not only provide health benefits but also taste good and eat well. Baking products that
provide for the good health of customers is an easily attainable goal for bakers.

Sucrose (Regular Refined Sugars)

Sucrose (commonly called sugar), a nonreducing disaccharide b-D-fructofuranosyl-a-D-

glucopyranoside) refined from sugar cane or beets, provides a clean sweetness in baked goods with
no off fivors. It is used either alone or in combination with other complementary sweetening
ingredients, and it is still the most common sweetener in baked goods. Refined sugars in bakery are
commonly classified by the size of the grains. The most commonly used are regular granulated sugar,
also called fine granulated or table sugar, and confectioners or powdered sugars. Granulated sugars
are further classified into ultra fine, very fine, and sanding sugars, depending on the size of the
grains. In general, finer granulations are better for mixing into doughs and batters because they
dissolve more quickly. Coarse sugars are likely to leave undissolved grains, even after long mixing.
These show up after baking as dark spots on crusts, irregular texture, and syrup spots. Also, fine
sugars are better for creaming with fats because they create a finer, more uniform air cell structure
and better volume. Coarse sugar, on the other hand, can be used in syrups, where its mixing
properties are not a factor.

Confectioner’s or powdered sugars are ground to a fine powder and mixed with a small amount of
starch (about 3%) to prevent caking. They are also classified by coarseness or fineness. 1OX is the
finest sugar. It gives the smoothest textures in icings. 6X is the standard confectioner’s sugar. It is
used in icings, toppings, and cream fillings. Coarser types (XXXX and XX) are used for dusting.

Invert Sugar

Liquid sucrose is available and is used primarily for yeast-leavened doughs. Invert sugar, made by
boiling sucrose with a dilute acid to break the sucrose into its constituent monomers, glucose and
fructose, is still popular in bakery goods. At 75% solids, the invert syrup is as sweet as sucrose. Invert
syrup is hydroscopic and resists crystallization; it is used where moisture absorption and retention
are important, as in fillings and icings. It is also brushed on Danish pastries after baking to help seal
in moisture.

Fondant

Fondant is sugar syrup that is crystallized to a smooth, creamy, white mass. When applied as icing, it
sets up into a shiny, nonsticky coating. Because it is difficult to make in the bakery, fondant is almost
always purchased already prepared, either in the ready-to-use moist form or in the dry form, which
requires only the addition of water. During the manufacture of fondant, part of the sucrose is
changed to invert sugar to get the right amount of crystallization. This helps keep the sugar crystals
very tiny, which makes a very smooth creamy icing with a good shine.

Brown Sugar

Brown sugar is mostly sucrose (about 85-92%), but it also contains varying amounts of caramel,
molasses, and other impurities, which give it its characteristic fivor. Basically, brown sugar is regular
cane sugar that has not been completely refined. However, it can also be made by adding measured
amounts of these impurities to refined white sugar. Its effects on crust and crumb color are related
to the darkness of the sugar-the more molasses, the darker the color. It also has softening
capabilities due to its solubility and the presence of the molasses, which contains reducing sugars.

Molasses

Molasses is concentrated sugar cane juice, containing large amounts of sucrose and other sugars
(including invert sugar). It also contains acids, moisture, and other constituents that give it its
characteristic flavor and color. Darker grades are stronger in fivor and contain less sugar than lighter
grades. Its main function in cookies is flavor, but molasses also adds humectancy due to the
presence of the reducing sugars glucose and fructose. Its natural acids may react with the leavening
system when combined with sodium bicarbonate (baking soda) and also affect the spread of cookies.

Fructose

Crystalline fructose b-D-fructofuranose) is another sweetener that may be used. It has sweetness
synergy with other sweeteners, including sucrose, which may allow cost savings. Because it is a
reducing sugar it can contribute too much color development if not used wisely. Fructose is sweeter
than sucrose, on a weight basis, and lowers water activity (aw) more effectively. It is more expensive
than sucrose and is normally used only in bar fillings where low aw, is critical.

Honey

Honey is a natural sugar syrup consisting largely of the simple sugars glucose and fructose, in
addition to other compounds that give it its flavor. It contains 41% fructose, 34% dextrose, 2%
sucrose, and 18% moisture. Honey varies widely in flavor and color based on the nectar source.
Honey has some great functional properties as a sweetener, but due to its high cost it is primarily
used for flavoring. Composed mostly of invert sugars, it acts as a humectant. Its reducing sugars
promote browning and increase spread. To lower cost, it is often found in blends with HFCS and
invert sugar syrups, which can be used as replacements when combined with honey flavors. Honey
also is available in free-flowing powders, fikes, granules, and crystals. Sometimes honey is used as
the sweetening agent in cakes. Actually, the use of honey in fiences more than simply the sweetness
of the cake because of its liquid content and acidity. Because honey is a liquid, it does not provide
crystals to aid in formation of a foam during creaming of the fat; thus, honey containing cakes tend
to be a bit coarser than those made with only granular sugar. The acidity is quite variable in different
honeys; between 1/12 and 1/2 teaspoon of soda may be needed to neutralize a cup of honey, but
the batter is permitted to remain more acidic in most cases. Honey promotes rapid browning
because of the abundance of reducing sugars it adds to the batter. Reducing sugars are very
susceptible to Maillard browning reactions. The high fructose content of honey is noteworthy in its
effect on browning.

Malt Syrup

Malt syrup, also called malt extract, is used primarily in yeast breads. It serves as food for the yeast
and adds fivor and crust color to the breads. Malt is extracted from barley that has been sprouted
(malted) and then dried and ground. There are two basic types of malt syrup, diastatic and non-
diastatic. Diastatic malt is produced with high, medium, or low diastase content. It is used when
dough fermentation times are short because diastase in malt breaks down starch into sugars that
can be a powerful food for yeast. It should not be used when fermentation times are long because
too much starch will be broken down by the enzyme, resulting in a sticky dough and crumb.
Nondiastatic malt is processed at high temperatures, which destroy the enzymes and give the syrup
a darker color and stronger fivor. It is used because it contains fermentable sugar and contributes
flavor, crust color, and keeping qualities to breads.

The use of baker’s yeast (Saccharomyces cerevisiae) as a leavening agent is a recent widely used
addition to bread making, dating back only 150 years (Batt and Tortorello, 2014). In contrast,
sourdough, which contains a culture of mostly wild yeast as well as lactic and acetic acid bacteria
that naturally inoculate bread dough, has been used as a leavening agent since ancient times
(Minervini et al., 2014). Sourdough fermentation releases several compounds that are not found in
modern yeast fermentation, and sourdough breads were shown to increase mineral bioavailability
(Arendt et al., 2007; Leenhardt et al., 2005; Lopez et al., 2003) and to induce advantageous effects
on glucose metabolism (Lappi et al., 2010).
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Lappi, J., Selinheimo, E., Schwab, U., Katina, K., Lehtinen, P., Mykkanen, H., Kolehmainen, M., and
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Leenhardt, F., Levrat-Verny, M.-A., Chanliaud, E., and Remesy, C. (2005). Moderate decrease of pH
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Lopez, H.W., Duclos, V., Coudray, C., Krespine, V., Feillet-Coudray, C., Messager, A., Demigne, C., and
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reconstituted whole wheat flour

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Saccharomyces cerevisiae, also referred to as baker’s yeast, is the primary leavening agent in the
production of bread in its numerous forms (Newberry and others 2002). Yeast cells consume the
fermentable sugars present in dough and generate carbon dioxide (CO2) and ethanol that are
responsible for dough leavening during the fermentation phase and the oven rise. Research has
shown that baker’s yeast serves other functions as well. Besides the 2 primary fermentation
metabolites, other metabolites such as organic acids, glycerol, and aroma compounds are produced.
These compounds

have an important impact on the bread making process and final bread quality (Cho and Peterson
2010; Jayaram and others 2013). Osmotic stress can strongly affect yeast’s fermentation rate
(Sasano and others 2012b).

Substrate preference of yeastin bread dough

Glucose and fructose are the preferred sugar sources of S. cerevisiae (Carlson 1999). Although
fructose is used concomitantly with glucose, consumption of the latter is faster when both sugars are
present (Berthels and others 2004). Glucose slows down the uptake of fructose. Indeed, both sugars
are imported into yeast cells by the same carriers, but they show a greater affinity for glucose than
for fructose (Verstrepen and others 2004).

When high levels of damaged starch (such as 8%) are present in the flour, maltose levels can reach
up to 6%. These high maltose levels can cause osmotic stress to yeast, leading to a lower yeast
viability and lower fermentation rates (Struyf and others 2017).

Sucrose is commonly used at varying levels in different

alcoholic fermentation systems for improvement of flavor and as a nutrient source for yeast cells
(Nagodawithana and Trivedi 1990). When sucrose concentrations are very high (contain sucrose
levels up to 30% yeast cells experience severe osmotic stress. This damages cellular components and
reduces their fermentation ability (Verstrepen and others 2004). Indeed, exposure of yeast cells to
hyperosmotic conditions leads to rapid cell dehydration and limits the cell growth and CO2
production capacity (Randez-Gil and others 2003). As a consequence of hyperosmotic pressure, the
fermentation rate and the final volume of the baked product are reduced (Myers and others 1997;
Hernandez-Lopez and others 2003)

(Fig.2)

Struyf N, van der Maelen E, Hemdane S, Verspreet J, Verstrepen KJ, and Courtin CM. 2017. Bread
Dough and Baker’s Yeast An Uplifting. Journal of Comprehensie Reviews in Food Science and Food
Safety Vol. 16. Insitute of Food Technologists. DOI: 10.1111.1541-4337.12282

Figure 2–Carbon sources of yeast cells during bread dough fermentation. Sources of fermentable
sugars are indicated in grey letters. In yellow, the structures of the different sources of fermentable
sugars are represented. In black letters, accompanied by scissors, enzymes are indicated. The upper
part of the figure (above the red line) represents the generation of fermentable sugars by
endogenous (wheat) or yeast enzymes. The lower part of the figure (under the red line) represents
the generation of (added) fermentable sugars by added or yeast enzymes.
Berthels N, Otero RC, Bauer F, Thevelein J, Pretorius I. 2004. Discrepancy in glucose and fructose
utilisation during fermentation by Saccharomyces cerevisiae wine yeast strains. FEMS Yeast Res
4:683–9.

Carlson M. 1999. Glucose repression in yeast. Curr Opin Microbiol 2:202–7

Cho IH, Peterson DG. 2010. Chemistry of bread aroma: a review. Food Sci Biotechnol 19:575–82

Hernandez-Lopez M, Prieto J, Randez-Gil F. 2003. Osmotolerance and leavening ability in sweet and
frozen sweet dough. Comparative analysis between Torulaspora delbrueckii and Saccharomyces
cerevisiae baker’s yeast strains. Antonie Leeuwenhoek 84:125–34.

Jayaram VB, Cuyvers S, Lagrain B, Verstrepen KJ, Delcour JA, Courtin CM. 2013. Mapping of
Saccharomyces cerevisiae metabolites in fermenting wheat straight-dough reveals succinic acid as
pH-determining factor. Food Chem 136:301–8

Myers D, Lawlor D, Attfield P. 1997. Influence of invertase activity and glycerol synthesis and
retention on fermentation of media with a high sugar concentration by Saccharomyces cerevisiae.
Appl Environ Microbiol 63:145–50

Nagodawithana TW, Trivedi NB. 1990. Yeast selection for baking. In: Panchal CJ, editor.Yeast strain
selection. New York, N.Y., U.S.A.: Marcel Dekker, Inc, p 139–84

Newberry M, Phan-Thien N, Larroque O, Tanner R, Larsen N. 2002. Dynamic and elongation rheology
of yeasted bread doughs. Cereal Chem 79:874–9

Randez-Gil F, Aguilera J, Codon A, Rinc ´ on AM, Estruch F, Prieto JA. 2003. Baker’s yeast: challenges
and future prospects. In: de Winde JH, editor. Functional genetics of industrial yeasts. Berling,
Heidelberg: Spriner-Verlag. p 57–97

Sasano Y, Haitani Y, Ohtsu I, Shima J, Takagi H. 2012b. Proline accumulation in baker’s yeast
enhances high-sucrose stress tolerance and fermentation ability in sweet dough. Intl J Food
Microbiol 152:40–3.
Struyf N, Laurent J, Verspreet J, Verstrepen KJ, Courtin CM. 2017b. Substrate-limited Saccharomyces
cerevisiae yeast strains allow controlling fermentation during bread making. J Agric Food Chem.
65(16):3368–77.

Verstrepen KJ, Iserentant D, Malcorps P, Derdelinckx G, Van Dijck P, Winderickx J, Pretorius IS,
Thevelein JM, Delvaux FR. 2004. Glucose and sucrose: hazardous fast-food for industrial yeast?
Trends Biotechnol 22:531–7.

Leavening agents either chemical or biological are important in raising flour dough. Biological
leavening agents are microorganisms that can produce carbon dioxide from the utilization of sugar
[1]. Yeast plays an important role in various fermentation processes including baking and brewing. In
brewing, the alcohol released by the fungus during fermentation is important while carbon dioxide is
of utmost need for rising of flour dough, maturation and development of fermentation flavour [2].

Fermentation of sugars by yeast is the oldest application in the making of bread, beer and wine. The
Babylonians (6000BC) and Egyptians (5000BC) have left written accounts of their production of beer,
wines and bread, where all of them warranted the use of yeast [3]. Yeast especially Saccharomyces
cerevisiae is known as sugar-eating fungus and can be found naturally from the surrounding.
According to

Kurtzman and Fell [4], fruits, vegetables, drinks and other agricultural products are very important
microhabitats for several of yeast species. A succession of yeast populations in such products
involves in a variety of biochemical processes carried out by yeast to utilize simple sugars

present in the agricultural products.

Baker’s yeast refers to the strains of Saccharomyces cerevisiae used in the bakery industry [5]. The
strains are different from the laboratory strains in their DNA content and chromosomal
polymorphism; most of the industrial strains are aneuploids [6]. It required having several
characteristics such as high leavening ability, osmo-tolerance, freeze tolerance, chemical tolerance,
melibiose utilization, good storage ability, and nonagglomeration [7]

High concentration of ethanol is reported to be toxic to the yeast by inhibiting the cells growth due
to the destruction of the cell membrane [18]. This could have accounted for the inhibiting growth of
most of the yeast species at 15% concentration as recorded in this research. A suitable concentration
of ethanol is needed in bread making to achieve the preferred flavour [18].
Results of the research show that all strains were able to ferment sugars namely dextrose, sucrose
and fructose. Yeast species were ferment different sugars indicates that they may be used as starter
culture in bakery industries. In conclusion, the study reported in this paper indicates

that yeast species identified from local fruits are excellent

habitat where yeasts with potentials for baking industrial

application, particularly yeasts with dough leavening ability

as observed in this research. In all the attributes considered, the yeast species namely
Saccharomyces cerevisae,

Saccharomyces blourdeous, Zygosaccharomyces fermentatii, Candida sorboxylosa,

Zygosaccharomyce bisporus, Kluveromyces delphensis, Pichia holistii and Candida apicola had the
overall best performance compared to commercial yeast. It was evidence from these findings that
local fruits could be source for baker’s yeast which is potentially used as dough leavening agent
(Tsegaye et al, 2018).

Tsegaye Z, Tefera G, Gizaw B, Abatenh E. 2018. Characterization of Yeast Species Isolated from Local
Fruits used for Bakery Industrial Application. J Appl Microb Res Vol: 1, Issu: 1 (21-26)

1. El Sheikha AF and Montet D (2016) Fermented Foods Artisan Household Technology to

Biotechnology Era. Fermented Foods, Part I: Biochemistry and Biotechnology. CRC Press, pp. 1-15.

2. Balarabe MM, Sani SDM, Orukotan AA (2017) Screening of Fermentative Potency of Yeast Isolates
from Indigenous Sources for Dough Leavening. Int J Microbiol Biotechnol 2: 12-17.

3. Kevin K (2005) Fungal fermentation systems and products. John Wiley and Sons Ltd, England.

4. Kurtzman CP and Fell JW (1998) The Yeasts: A Taxonomic Study. Elsevier Science, Amsterdam.

5. Phaff HJ, Miller MW, Mrak EM (1978) Industrial uses of yeast. Harvard University Press,
Cambridge, MA.

6. Codon AC, Benitez T and Korhola M (1998) Chromosomal polymorphism and adaptation to specific
industrial environments of Saccharomyces strains. Appl Microbiol Biotechnol 49: 154-163.

7. Walker GM (1998) Baker’s yeast. Wiley and Sons, Chichester.

18.Stanley D, Bandara A, Fraser S, Chambers PJ, Stanley GA (2010) The ethanol stress response and
ethanol tolerance of Saccharomyces cerevisiae. J Appl Microbiol 109: 13-24..
https://doi.org/10.1111/j.1365-2672.2009.04657.x

PEOPLE AROUND THE world are again becoming fascinated by sourdoughs. Why the fascination? The
simple answer is that, in many ways, sourdoughs improve the quality of our lives. They have a
unique, inherent charisma, and they still produce the best bread the world has ever known. They are
soul-satisfying and fun: we call them “endorphins of the kitchen.” They truly offer an extension of a
personal quality beyond what we eat to what we do and what we are.

We know the sourdough process results from the fermentation reactions of two quite different
classes of microorganisms:

wild yeast and beneficial bacteria. For well over five thousand years, all breads were produced by
the fermentation of these two essential microorganisms acting together. The yeasts are primarily
responsible for leavening and bread texture, the bacteria for the sourdough flavor. Thus the
definition of “traditional” sourdough requires a “culture,” or “starter,” containing both of these
organisms.

Bakers of every sort welcomed the introduction of commercial yeast in the late 1800s. It greatly
simplified the baking process and made it much faster. But something happened to the sourdough
flavor. It disappeared! In due time, researchers identified the problem. They found that sourdough
process results from the fermentation reactions produced not only one microorganism but two quite
different classes of microorganisms: wild yeasts make it rise and beneficial bacteria provide the
flavor.

Wood E, Wood J. 2011. Classic Sourdoughs: A Home Baker's Handbook (Revised 4th). Ten Speed
Press: Barkeley-USA.

Wild-type yeasts possess characteristic aroma and assimilative ability associated with the growth
environments

such as flowers, fruits, leaves, and branches. Recently, development of breads using wild-type yeast
is receiving

a lot of attention in baking industries (Shimosaka, 2011)

Shimosaka, C. (2011). Preparation method and puffing behavior of bread with Shirakamikodama
yeast. Journal of Cookery Science of Japan, 44, 223-230.
https://doi.org/10.11402/cookeryscience.44.223
Beer, wine and bread have been produced with the help of microorganisms for thousands of years.
Yeast has been found to be responsible for the occurrence of alcoholic fermentation, i.e., the
production of ethanol and carbon

dioxide from fermentable sugars during brewing, wine making and dough leavening.

The “microbial theory” of alcoholic fermentation evoked a remarkable attack by some leading
chemists for a long period of time [17]. It took about 20 years until Pasteur’s study led, once and for
all, to the general acceptance of the fact that alcoholic fermentation was a process performed by
living organisms [18]

It is strongly assumed that yeast strains which are currently used in these industries are
domesticated due to their long-term association with humaninduced fermentation. Nevertheless, it
is still controversial whether S. cerevisiae strains occasionally occurring in nature are derived from
domesticated strains or vice versa [1, 2]. Indeed, there is some thought that all S. cerevisiae strains
originally derive from close relatives occurring in nature. However, several recent genomic studies
seem to invalidate this hypothesis by identifying natural S. cerevisiae populations not associated
with human alcoholic

beverage production [3].

According to recent taxonomic nomenclature, virtually all industrially used baker’s, wine and
brewer’s yeast strains belong to the Saccharomyces sensu stricto species [30, 31]. Baker’s yeast,
many wine starter yeast strains and top-fermenting brewer’s yeast (ale yeast) have been assigned to
the species Saccharomyces cerevisiae [32–35]. Bottom-fermenting (lager) brewer’s yeast originally
known as S. carlsbergensis according to Hansen [39] were later recognized as part of the S.
pastorianus species [40]. In terms of their genetic constitution, bottom-fermenting (lager) brewer’s

yeast are most striking among industrial yeast strains.

Increasing Glycerol Formation for Improved Wine Viscosity

and in Order to Reduce the Ethanol Content in Alcoholic Beverages

Glycerol affects the sensory quality of wine, due to its nonvolatile character and slightly sweet taste.
Therefore, a moderate increase in glycerol content is desired; it improves viscosity, sweetness,
consistency and the overall body of

wine [25]. Glycerol overproduction has been also considered a valuable strategy to produce
beverages with decreased ethanol content, as any increase in glycerol production will
simultaneously lead to a decrease in ethanol yield. Consumer demand for low-ethanol beer and
wine is indeed continuously increasing due to both increased awareness for health and stricter laws
regarding drinking and driving. Glycerol in S. cerevisiae is produced by the reduction of
dihydroxyacetone

phosphate, catalyzed by the glycerol-3-phosphate dehydrogenase (GPDH, Fig. 3). As glycerol

overproduction has also been of interest in industrial biotechnology, there have been several
approaches to overproduce glycerol

in laboratory strains of S. cerevisiae [157]. The first strategy to improve glycerol production in
brewer’s and wine

yeasts was the overproduction of glycerol 3-phosphate dehydrogenase (GPD), which is the rate-
controlling step in glycerol biosynthesis [161–163].

The high production of acetate has been the main disadvantage in wine yeast. A novel avenue to
reduce ethanol in beverages could be the use of yeast strains with an abolished Crabtree effect.
Based on the approach of Elbing et al. [167], a nonethanol-producing wine yeast strain was
developed by modification of hexose transporters [168]. Moreover, introducing heterologous
enzymes leading to increased NADH oxidation has been shown to reduce overflow metabolism, i.e.,
ethanol formation, in wine yeast [169]. As the functionality of such approaches relies on oxygen
availability or, at least, microaeration, their use for production of low-alcohol beverages will require
sophisticated fermentation strategies. Indeed, full aeration would strongly change yeast’s
metabolism and byproduct formation in comparison to traditional beer and wine production carried
out under oxygen-limited conditions.

Improving Tolerance to Several Types of Stress

Expression studies of suitable marker genes revealed osmotic and oxidative stress as the major
causes of stress response under such conditions [178]

Fig. 3 Glycerol, ethanol and acetate production linked to the glycolytic pathway in S. cerevisiae.
Genes and enzymes discussed in this review: GPD1 and GPD2 isogenes encoding glycerol-3-
phosphate dehydrogenase, GPP1 and GPP2 isogenes encoding glycerol3-phosphatase, ALD2/3/6
genes encoding cytosolic acetaldehyde dehydrogenase, FPS1 encoding a channel protein which
facilitates glycerol transport through the plasma membrane (Donalies, 2008).

Donalies U.E.B., Nguyen H.T.T., Stahl U., Nevoigt E. 2008. Improvement of Saccharomyces Yeast
Strains Used in Brewing, Wine Making and Baking. J.Adv Biochem Engin/Biotechnol 111: 67–98. DOI
10.1007/10_2008_099

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32. Barnett JA (1992) Yeast 8:1

33. Pretorius IS (2003) In: De Winde JH (ed) Functional genetics of industrial yeasts. Springer, Berlin
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34. Hansen J, Kielland-Brandt MC (2003) In: De Winde JH (ed) Functional genetics of industrial
yeasts. Springer, Heidelberg, p 143

35. Randez-Gil F, Aguilera J, Codon A, Rincon AM, Estruch F, Prieto JA (2003) In: de Winde JH (ed)
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39. Hansen E (1908) Comp Rend Trav Lab Carlsberg 7:179

40. Martini AV, Martini A (1987) Antonie Van Leeuwenhoek 53:77

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163. Michnick S, Roustan JL, Remize F, Barre P, Dequin S (1997) Yeast 13:783

167. Elbing K, Larsson C, Bill RM, Albers E, Snoep JL, Boles E, Hohmann S, Gustafsson L (2004) Appl
Environ Microbiol 70:5323

168. Henricsson C, De Jesus Ferreira MC, Hedfalk K, Elbing K, Larsson C, Bill RM, Norbeck J, Hohmann
S, Gustafsson L. 2005. Appl Environ Microbiol 71:6185

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Efektivitas nira aren sebagai bahan pengembangan adonan roti lebih rendah daripada menggunakan
bahan pengembang ragi instan. Semakin panjang umur nira aren (semakin lama nira aren disimpan)
semakin rendah efektivitasnya terhadap

pengembangan adonan dan semakin rendah kualitas roti yang dihasilkan.

Sehubungan dengan rendahnya efektivitas nira aren sebagai bahan, maka bahan tersebut kurang
baik digunakan sebagai bahan pengembang adonan roti akan tetapi dapat digunakan sebagai bahan
pengembang adonan cake yang membutuhkan pengembangan yang tidak terlalu besar.

Lempang M, Mangopang AD. 2012. Efektivitas Nira Aren Sebagai Bahan Pengembang Adonan Roti.
Jurnal Penelitian Kehutanan Wallacea Vol 1 No.1 Agustus 2012:26-35.

The baker’s yeast is more sensitive to alcohol concentration compared with either brewer’s or wine
yeasts (Ali Shah, 2010). Prior to the end of sponge fermentation the decline of yeast activity is to be
subjected to the rising of alcohol content of the dough than to a depletion of yeast food. Satisfied
fermented sponge dough is obtained at 3% of ethanol based on flour. It has been observed that
alcohol concentration depresses yeast fermentation activity about 20% prior to the end of
fermentation. (Pyler, 1988).

Ali Shah SF. 2010. Enhanced Production of Ethanol From Sugar Cane Molasses Through
Thermotolerant Saccharomyces cerevisiae cell. DOI:10.13140/RG.2.1.3418.8968
Pyler, E. J. (1988). Baking Science and Technology . 3rd ed. Sosland Publishing Company, Merriam,
Kansas. U.S.A

Microbes Viability, pH, Alcohol Level, Volubility and Sweet Bread Dough Gas Volume, in Fresh Yeast
Production Used Soursop as Raw Material.

Wahidah N. 2016. Viabilitas Mikroba, pH, Kadar Alkohol, Daya Kembang, dan Volume Gas Adonan
Roti Manis pada Proses Pembuatan Yeast Segar Berbahan Dasar Sirsak. Skripsi: Universitas
Diponegoro.

Hasil penelitian yang telah dilakukan menunjukkan bahwa

terdapat perbedaan nyata (p<0,05). Pada hasil viabilitas yeast menunjukkan 0,00-9,56 CFU/ml; nilai
pH menunjukkan hasil 6,29-4,05 ; kadar alkohol menunjukan hasil 0,00-7,22% ; daya kembang
adonan menunjukkan hasil 0,00-2,36 cm3; dan volume gas yang dihasilkan menunjukkan hasil 0,00-
108,75 ml. Sehingga dapat ditarik kesimpulan bahwa semakin lama fermentasi yang dilakukan pada
yeast segar buah sirsak mempengaruhi peningkatan viabilitas mikroba, kadar alkohol,

daya kembang adonan roti manis, dan volume gas yang dihasilkan. Namun lama fermentasi yang
dilakukan pada yeast segar menyebabkan penurunan nilai pH yeast buah sirsak. Penelitian ini dapat
disimpulkan bahwa dari 5 perlakuan yang diujikan hasil fermentasi optimal terjadi pada lama
fermentasi 96 jam.

BAB I PENDAHULUAN

1.1. Latar Belakang

1.2. Tujuan dan Manfaat

1.3. Hipotesis

BAB II TINJAUAN PUSTAKA

2.1. Yeast

2.2. Yeast Segar

2.3. Proses Pembuatan Yeast Segar

2.4. Sirsak
2.5. Adonan Roti

2.6. Viabilitas Mikroba

2.7. Nilai pH

2.8. Kadar Alkohol

2.9. Daya Kembang Adonan Roti

2.10. Volume yang Dihasilkan

BAB III MATERI DAN METODE

3.1. Materi Penelitian

3.2. Metode Penelitian

BAB IV HASIL DAN PEMBAHASAN

4.1. Viabilitas Mikroba

4.2. Nilai pH

4.3. Kadar Alkohol

4.4. Daya Kembang Adonan Roti Manis

4.5. Volume Gas yang Dihasilkan

4.6. Viabilitas Mikroba, Nilai pH,Kadar Alkohol, Daya Kembang

Adonan Roti Manis, dan Volume Gas yang Dihasilkan

BAB V SIMPULAN DAN SARAN

5.1. Simpulan

5.2. Saran

DAFTAR PUSTAKA

LAMPIRAN

RIWAYAT HIDUP

PENDAHULUAN

1.1. Latar Belakang

Perkembangan dunia bakery di Indonesia semakin melaju pesat, berbagai macam inovasi roti terus
dikembangkan karena adanya tuntutan dari pasar yang menginginkan varian roti yang selalu
bervariasi. Namun tidak disadari karena tuntutan inovasi dan kebutuhan masyarakat terhadap
konsumsi roti yang terus meningkat menyebabkan produksi roti banyak menggunakan bahan
tambahan pangan buatan. Bahan tambahan pangan buatan ditambahkan untuk mempercepat
proses produksi. Bahan tambahan yang digunakan sebagian besar adalah bahan pengawet agar roti
yang diproduksi tidak cepat rusak karena adanya penurunan mutu secara fisik maupun kimia dan
juga bahan pengembang instan untuk mempercepat pengembangan roti. Banyak masyarakat yang
tidak menyadari penggunaan bahan tambahan kimia pada produk pangan akan menimbulkan
pengendapan residu bahan kimia dalam tubuh yang dapat mengganggu kesehatan karena tubuh
akan kesulitan mencerna residu tersebut.

Penggunaan bahan pengembang dalam pembuatan roti sangat penting untuk menghasilkan roti
yang diinginkan. Roti yang dihasilkan di skala industri biasanya menggunakan bahan pengembang
buatan. Bahan pengembang buatan dipilih karena dapat mengembangkan roti dengan cepat dan
dengan biaya yang relatif murah. Kualitas roti yang baik dapat dilihat dari tingkat daya kembang dan
tekstur roti yang dihasilkan. Yeast merupakan suatu bahan yang tidak dapat dipisahkan dalam proses
pembuatan adonan roti. Salah satu bahan yang mempengaruhi kualitas roti adalah yeast yang
digunakan. Menurut Mulyono (2011) fungsi utama yeast adalah mengembangkan adonan roti. Saat
proses pengembangan adonan terjadi yeast akan menghasilkan gas karbondioksida (CO2) selama
fermentasi. Karbondioksida kemudian terperangkap dalam jaringan gluten yang menyebabkan roti
dapat mengembang. Komponen lain yang terbentuk selama proses fermentasi adalah asam dan
alkohol yang berkontribusi terhadap rasa dan aroma roti, namun alkohol akan menguap dalam
proses pengembangan roti. Proses fementasi adalah salah satu hal paling penting dalam pembuatan
adonan roti. Salah satu bahan baku roti yang paling penting dalam proses pembuatan roti adalah
ragi atau yeast. Yeast adalah mikroorganisme hidup yang berkembang biak dengan cara memakan
gula (Pratama et al., 2015). Viabilitas mikroba sangat mempengaruhi terhadap pengembangan
adonan dan produksi gas yang akan terjadi saat fermentasi.

Proses pengembangan adonan merupakan suatu proses terjadinya sinkronisasi antara peningkatan
volume sebagai akibat bertambahnya gas-gas yang terbentuk sebagai hasil fermentasi dan protein
larut, lemak dan karbohidrat yang mengembang. Yeast yang selama ini digunakan oleh masyarakat
pada umumnya adalah dry instant yeast karena kemampuannya untuk mengembangkan adonan roti
yang sangat cepat, mudah ditemukan dipasaran dan harganya yang terjangkau.

Namun pada penelitian yang telah dilakukan oleh seorang pakar bakery asal Korea Selatan yang
bernama Sangjin Ko, dan penelitiannya telah dibukukan dengan buku berjudul Rahasia Membuat
Roti Sehat dan Lezat dengan Ragi alami membuktikan bahwa yeast untuk mengembangkan adonan
dapat diperoleh dari bahan-bahan alami seperti sayuran, buah dan serealia dengan syarat bahan
yang difermentasi memiliki cukup karbohidrat yang nantinya akan diubah menjadi karbondioksida
dan alkohol.

Yeast segar adalah yeast yang didapat dari fermentasi berbagai macam bahan pangan yang
mengandung karbohidrat dapat diperoleh dari buah-buahan, sayuran dan serealia. Mikroorganisme
yang dihasilkan dari proses fermentasi yeast segar sangat beragam tergantung bahan yang
digunakan untuk fermentasi. Mikroorganisme yang teridentifikasi pada yeast segar antara lain
Saccaromyces cerevisae, Saccaromyces ellipsoideus, Saccaromyces bayanus, Saccaromyces buchneri,
Saccaromyces exiguos (Ko, 2012).

Penggunaan yeast segar banyak digunakan oleh ahli bakery luar negeri seperti di Prancis, Korea,
Eropa dan negara- negara lain karena penambahan yeast alami pada adonan roti akan membuat roti
yang dihasilkan memiliki tekstur yang lembut dan memiliki bau khas (Ko, 2012). Amilase dan
protease yang terkandung dalam proses pembuatan roti ini dapat membantu memecah beberapa
gluten. Sehingga roti jenis ini lebih mudah dicerna oleh orang-orang yang intoleran terhadap gluten.
Di Indonesia sendiri penggunaan yeast segar belum banyak diterapkan. Penggunaan yeast di
Indonesia biasanya dipakai dalam pembuatan roti untuk para vegetarian. Buah sirsak adalah salah
satu buah lokal yang dapat dimanfaatkan sebagai salah satu alternatif bahan dasar dalam proses
pembuatan yeast segar karena kandungan karbohidratnya yang cukup tinggi dan memiliki pH asam.
Buah sirsak pada umumnya hanya dikonsumsi begitu saja ataupun dijadikan jus buah. Pengunaan
buah sirsak dalam pembuatan yeast segar adalah salah satu modifikasi pangan yang diharapkan
dapat meningkatkan nilai tambah pada buah sirsak.

1.2. Tujuan dan Manfaat

Penelitian ini bertujuan untuk mengoptimalkan sirsak sebagai bahan yeast segar dan menganalisis
hasil yeast segar buah sirsak dengan uji viabilitas mikroba, volume pengembangan adonan,
pengukuran nilai pH, produksi gas yang dihasilkan dan volume alkohol adonan roti manis pada
proses pembuatan yeast segar berbahan dasar sirsak dengan berbagai lama waktu fermentasi
sehingga dapat diketahui waktu optimal pada fermentasi yeast segar berbahan dasar sirsak.

Manfaat yang diharapkan dari penelitian ini adalah dapat mengoptimalkan sirsak sebagai yeast segar
sehingga dapat dijadikan salah satu alternatif penggunaan yeast dalam proses pembuatan adonan
roti. Selain itu dapat meningkatkan nilai tambah pada sirsak dengan adanya modifikasi pangan
tersebut.

1.3. Hipotesis

Hipotesis penelitian terdapat pengaruh penambahan yeast segar buah sirsak terhadap viabilitas
mikroba, pH, kadar alkohol, volume daya kembang adonan dan volume gas yang dihasilkan pada
proses pembuatan adonan roti manis.

BAB II

TINJAUAN PUSTAKA

2.1. Yeast

Yeast merupakan mikroorganisme yang termasuk dalam fungi uniseluler yang menyebabkan
terjadinya fermentasi. Yeast biasanya mengandung mikroorganisme yang melakukan fermentasi dan
media biakan bagi mikroorganisme tersebut. Media tumbuh yeast ini dapat berbentuk cairan
nutrien. Yeast umumnya digunakan dalam industri pangan untuk membuat makanan dan minuman
hasil fermentasi seperti acar , roti dan bir. Yeast berkembang biak dengan suatu proses yang dikenal
dengan istilah pertunasan, yang menyebabkan terjadinya peragian. Dalam pembuatan adonan roti,
sebagian besar yeast berasal dari mikroorganisme jenis Saccharomyces cerevisiae. Yeast merupakan
bahan pengembang adonan dengan memproduksi gas karbondioksida (Mudjajanto dan Yulianti
2004).

Menurut US.Wheat Assosiates (1981) yeast terdiri dari sejumlah kecil enzim, termasuk protease,
invertase, maltase dan zymase. Enzim yang penting dalam yeast adalah invertase, maltase dan
zymase. Enzim invertase dalam yeast bertanggung jawab terhadap awal aktivitas fermentasi. Enzim
ini mengubah gula (sukrosa) yang terlarut dalam air menjadi gula sederhana yang terdiri atas glukosa
dan fruktosa. Gula sederhana kemudian dipecah menjadi karbondioksida dan alkohol. Enzim amilase
yang terdapat dalam tepung mampu memproduksi maltose yang dapat dikonsumsi oleh yeast
sehingga fermentasi terus berlangsung. Enzim zymase mengubah invert sugar dan dekstrosa menjadi
gas karbondioksida yang akan menyebabkan adonan menjadi mengembang dan terbentuk alkohol.
Enzim zymase merupakan biokatalis yang digunakan dalam proses pembuatan roti. Enzim zymase
dapat mengubah glukosa dan fruktosa menjadi CO2 dan alkohol. Penambahan enzim zymase
dilakukan pada proses peragian pengembangan adonan roti. Yeast ditambahkan kedalam adonan
roti sehingga glukosa dalam adonan roti akan terurai menjadi etil alkohol dan karbondioksida. Proses
penguraian ini berlangsung dengan bantuan enzim zymase terdapat pada yeast.

Yeast yang sering digunakan masyarakat dalam pembuatan adonan roti umumnya jenis instant dry
yeast yang pemakaiannya langsung dicampurkan dengan bahan lainnya. Menurut Mudjajanto dan
Lilik (2004), penggunaan yeast 1,5 – 2 % dari total tepung terigu. Selain menggunakan instant dry
yeast yang dijual dipasaran, dalam pembuatan adonan roti dapat pula menggunakan alternatif yeast
segar yang diperoleh dari fermentasi bahan-bahan organik yang mengandung karbohidrat seperti
buah-buahan, sayur-sayuran dan juga serealia. Ketika bahan pangan yang mengandung karbohidrat
dibiarkan didalam suhu ruangan dalam beberapa hari maka bahan pangan tersebut akan mengalami
perubahan secara enzimatis dan akan berubah menjadi asam (berbau seperti alkohol). Hal tersebut
membuktikan bahwa disetiap bahan pangan yang memiliki kandungan karbohidrat memiliki
kandungan yeast dan berpotensi dijadikan alternatif dalam pembuatan yeast alami (Ko, 2012).

2.2. Yeast Segar

Yeast segar adalah mikroorganisme dari bahan-bahan alami yang didapatkan dari hasil fermentasi
kultur alami dengan menangkap mikroorganisme alami yang terdapat dalam suatu bahan pangan.
Mikroorganisme dalam bahan-bahan alami menggunakan glukosa serta memproduksi
karbondioksida, aroma alkohol, dan asam-asam organik menggunakan mikroorganisme bermanfaat
yang berasal dari bahan-bahan alami.

Yeast segar diperoleh dari fermentasi buah-buahan, sayuran dan serealia yang memiliki jumlah
karbohidrat yang cukup . Yeast dalam buah akan memecah karbohidrat menjadi karbondioksida dan
alkohol. Penambahan yeast segar pada pembuatan adonan roti bermanfaat mempermudah daya
cerna roti karena pada saat fermentasi mikroorganisme mengubah senyawa yang kompleks menjadi
lebih sederhana, tekstur adonan akan lebih empuk dan lembut, karena berbagai macam
mikroorganisme dapat menghasilkan pelembab seperti trehalose yang dapat menghambat
retrogradasi pati pada roti sehingga keempukan roti menjadi lebih tahan. Umur simpan yang
panjang tanpa pengawet. Yeast segar kaya akan rasa dan aroma. Selama proses fermentasi, berbagai
metabolit dari mikrooganisme memberikan rasa dan aroma yang unik dan beragam (Ko, 2012).

Bahan yang digunakan dalam proses pembuatan yeast segar harus memiliki karbohidrat yang cukup
sehingga dapat diubah menjadi gula dan akan menghasilkan karbondioksida dan alkohol yang
nantinya akan digunakan untuk mengembangkan adonan roti manis. Selain itu bahan yang
digunakan dalam pembuatan yeast alami disarankan adalah bahan pangan yang memiliki sifat asam,
dikarenakan apabila bahan yang digunakan memiliki sifat basa hal tersebut akan memungkinkan
bakteri lain akan ikut tumbuh dan dapat mengkontaminasi yeast segar. Kismis adalah salah satu
buah yang memiliki kadar karbohidrat yang tinggi dan memiliki sifat asam sehingga dapat dijadikan
alternatif bahan untuk membuat yeast segar. Kandungan karbohidrat pada kismis bila difermentasi
akan menghasilkan yeast segar yang menghasilkan gas karbondioksida dan alkohol yang akan
dimanfaatkan untuk mengembangkan adonan roti manis. Selain itu kismis memiliki kandungan
antioksidan dan serat pangan yang tinggi sehingga baik untuk kesehatan.

2.3. Proses Pembuatan Yeast Segar

Prinsip dari pembuatan yeast segar adalah dengan fermentasi sederhana. Fermentasi adalah
perubahan kimia dalam bahan pangan yang disebabkan oleh enzim, enzim yang berperan dapat
dihasilkan oleh mikroorganisme atau telah ada dalam bahan pangan itu sendiri. Perubahan yang
terjadi sebagai hasil fermentasi mikroorganisme dan interaksi yang terjadi di antara produk dari
kegiatan-kegiatan tersebut dan zat-zat yang merupakan bahan pangan tersebut (Buckle, 2007).
Fermentasi merupakan suatu reaksi pengubahan glukosa menjadi alkohol dan karbondioksida. Saat
proses fermentasi terjadi pemecahan substrat oleh enzim-enzim tertentu terhadap bahan yang tidak
dapat dicerna seperti selulosa dan akan diubah menjadi gula sederhana. Selain itu pada saat
fermentasi berlangsung akan tumbuh jamur yang menghasilkan protein hasil metabolisme sehingga
dapat meningkatkan jumlah protein yang dikandung (Sembiring, 2013).

Semakin lama fermentasi dilakukan maka akan terjadi penurunan pH yang terus menerus, karena
pada saat fermentasi akan mengalami proses biosintesis piruvat yang menghasilkan produk asam
sehingga membuat pH akan terus menerus menurun dan menjadi asam (Utama dan Mulyanto,
2009). Selain berpengaruh pada pH, lama fermentasi juga berpengaruh pada alkohol yang dihasilkan,
semakin lama fermentasi Saccharomyces cerevisae berkembang biak akan bertambah
kemampuannya untuk memecah substrat atau glukosa menjadi alkohol (Kunaepah, 2008).

Reaksi kimia fermentasi :

C6H12O6 → 2C2H5OH + 2CO2 + 2 ATP

Pada saat proses fermentasi terjadi peningkatan panas, untuk meminimalisir panas yang dihasilkan
maka perlu dilakukan pendinginan agar suhu tetap stabil pada suhu 30oC selama proses fermentasi
(Santi, 2008). Pemanfaatan proses fermentasi pada bidang pangan sangat beragam. Fermentasi
digunakan sebagai salah satu usaha untuk meningkatkan nilai suatu bahan pangan dan juga untuk
mengawetkan bahan pangan.

Bahan pangan yang memiliki kandungan karbohidrat bila dibiarkan dalam ruangan terbuka dalam
jangka waktu yang cukup lama akan menyebabkan bahan pangan tersebut berbau asam (seperti
alkohol) hal tersebut menunjukkan bahwa dalam bahan pangan yang mengandung karbohidrat
memiliki kandungan yeast yang dapat dimanfaatkan sebagai yeast alami yang dapat diaplikasikan
dalam pembuatan adonan roti.

Pembuatan yeast segar dimulai dengan memilih bahan yang akan digunakan, mensterilisasikan alat,
pembuatan yeast segar (starter), pembuatan yeast induk dari starter yeast segar buah sirsak (Ko,
2012). Bahan yang digunakan harus memiliki kandungan karbohidrat yang cukup dan pH yang asam.
Karbohidrat akan dipecah menjadi gula yang akan diubah menjadi alkohol dan karbondioksida.
Sedangkan bahan pangan yang memiliki pH asam akan menguntungkan saat proses pembuatan
yeast segar karena pada pH asam mikroorganisme lain tidak dapat tumbuh dan mengkontaminasi
yeast.

2.6. Viabilitas Mikroba

Yeast segar dapat bertahan hidup pada suhu 25oC-27oC. Penyimpanan yeast segar harus dilakukan
pada suhu yang stabil, bila suhu ruangan fluktuatif akan mengakibatkan yeast tidak stres dan mati.
Penyimpanan yeast yang dianjurkan adalah memasukkannya kedalam box styrofoam untuk menjaga
agar suhu tetap stabil. Salah satu tanda yeast segar bertahan hidup saat fermentasi akan
menimbulkan gelembung-gelembung udara. Nutrisi yeast pada saat fermentasi harus dipastikan
tercukupi agar yeast tetap hidup dan berkembangbiak (Ko, 2012).

Uji viabilitas mikroba adalah pengujian yang dilakukan untuk mengetahui sel yeast yang masih hidup
dan yang mati. Pengujian viabilitas mikroba dengan menggunakan metode TPC (Total Plate Count)
dengan menggunakan media NA (Nutrient Agar). Pengujian dengan menggunakan menggunakan
media NA adalah pengujian yang lazim digunakan untuk pengujian mikrobiologis pada pangan
(Indriati et al., 2010). Prinsip perhitungan pengujian TPC ini dengan melakukan pengenceran,
pencawanan, dan perhitungan jumlah yeast yang masih bertahan hidup (Anal dan Singh, 2007).

2.7. Nilai pH

Menurut Ko (2012) nilai pH dalam proses fermentasi yeast segar sangat berperan penting dalam
aktivitas perkembangbiakan yeast karena akan mempengaruhi aktivitas enzim. Nilai pH optimal
yeast segar yang dianjurkan adalah pH 4-6. Sifat asam akan mencegah tumbuhnya bakteri lain
tumbuh yang berpotensi mengkontaminasi yeast segar. Peningkatan jumlah mikroba dipengaruhi
nilai pH, seiring penurunan pH aktivitas metabolisme pun akan meningkat sehingga semakin lama
fermentasi berlangsung nilai pH akan menurun (Koroleva, 1991).

Metode pengukuran nilai pH menggunakan pH meter. Semua sampel yang telah dihomogenisasi
diukur nilai pHnya. Pengukuran dilakukan dua kali (duplo) untuk menguji keakuratan nilai pHnya
selanjutnya diambil rata-ratanya. Nilai pH selama proses fermentasi semakin lama semakin turun,
hal ini disebabkan karena produksi asam sitrat, asam asetat dan isoasetat semakin lama semakin
meningkat sehingga kadar pH selama proses fermentasi semakin turun (Kayagil, 2006).

Penggunaan pH meter harus diperhatikan untuk mengkalibrasi pH meter sebelum digunakan untuk
mengukur pH suatu bahan agar didapatkan nilai pH yang akurat. Teknik kalibrasi yang akan
digunakan adalah teknik dua titik. Teknik dua titik adalah dengan menggunakan 2 buffer standart
yaitu pH 4,00 dan pH 7,00 (Tahir, 2008). Pengukuran pH dilakukan dengan mengkalibrasi pH meter
dengan buffer standar pH 4,00 dan pH 7,00 kemudian mengambil sampel yeast alami sebanyak 10ml
dan ditambahkan dengan 90 ml aquades kemudian dihomogenisasikan dan diukur pH yeast
tersebut.

2.8. Kadar Alkohol

Alkohol adalah hasil utama dari fermentasi, reaksi yang terjadi saat pembentukan alkohol terjadi
pada kondisi anaerob (Kwartiningsih dan Mulyati, 2005). Menurut Hemiarti (2012fMuy) salah satu
metode untuk mengkonversi karbohidrat menjadi alkohol adalah dengan metode fermentasi dan
metode sakarifikasi. Fermentasi akan mengubah molekul karbohidrat menjadi dua molekul alkohol
dan dua molekul karbondioksida. Untuk mendapatkan kadar alkohol yang tinggi maka harus
dilakukan proses destilasi (Fessenden dan Fesenden, 2001).

Alkohol adalah hasil samping dari fermentasi selain asam asetat, gliserol, dan asetaldehit
(Kwartiningsih dan Mulyati, 2005). Enzim invertase akan mengubah karbohidrat menjadi gula yang
lebih sederhana. Gula sederhana akan diubah menjadi alkohol dan karbondioksida dengan bantuan
enzim zymase.

Prosedur pengujian kadar alkohol dilakukan dengan metode piknometer sesuai dengan pendapat
Putri dan Sukandar (2008), pertama-tama sampel sebanyak 100 ml dimasukkan ke dalam labu
destilasi Kjeldahl kemudian ditambahkan dengan aquades sebanyak 100 ml. Selanjutnya didestilasi
pada suhu 80 C. Destilat ditampung di dalam erlenmeyer hingga volume 80 ml. Destilat tersebut
kemudian dimasukkan ke dalam piknometer berukuran 10 ml yang telah ditimbang sebelumnya.
Destilat dimasukkan hingga memenuhi piknometer. Piknometer yang berisi destilat ditimbang dan
beratnya dicatat. Prosedur yang sama dilakukan pada aquades sebagai pembanding. Hasil
penghitungan berat jenis alkohol kemudian dikonversikan dengan menggunakan tabel konversi
berat jenis alkohol.

Rumus berat jenis alkohol :

𝑏𝑒𝑟𝑎𝑡 𝑗𝑒𝑛𝑖𝑠 ∶ (𝑏𝑒𝑟𝑎𝑡 𝑝𝑖𝑘𝑛𝑜𝑚𝑒𝑡𝑒𝑟 + 𝑑𝑒𝑠𝑡𝑖𝑙𝑎𝑡) − 𝑏𝑒𝑟𝑎𝑡 𝑝𝑖𝑘𝑛𝑜𝑚𝑒𝑡𝑒𝑟 𝑘𝑜𝑠𝑜𝑛𝑔 / (𝑏𝑒𝑟𝑎𝑡 𝑝𝑖𝑘𝑛𝑜𝑚𝑒𝑡𝑒𝑟 +
𝑎𝑘𝑢𝑎𝑑𝑒𝑠) − 𝑏𝑒𝑟𝑎𝑡 𝑝𝑖𝑘𝑛𝑜𝑚𝑒𝑡𝑒𝑟 𝑘𝑜𝑠𝑜𝑛𝑔

3.2. Metode Penelitian

Metode penelitian meliputi rancangan percobaan, uji variabel penelitian, prosedur penelitian dan
analisis data. Uraian berikut menjabarkan tiap sub-sub bab tersebut.

3.2.1. Racangan Percobaan

Metode yang digunakan pada penelitian ini adalah metode eksperimen dengan menggunakan
monofaktor Rancangan Acak Lengkap (RAL) dengan variasi waktu fermentasi pada yeast alami
berbahan dasar buah sirsak T0 : penambahan yeast segar buah sirsak pada adonan roti sebesar 50%
dengan lama fermentasi yeast segar buah sirsak selama 0 jam, T1 : penambahan yeast segar buah
sirsak pada adonan roti sebesar 50% dengan lama fermentasi yeast segar buah sirsak selama 24 jam,
T2 : penambahan yeast segar buah sirsak pada adonan roti sebesar 50% dengan lama fermentasi
yeast segar buah sirsak selama 48 jam, T3 : penambahan yeast segar buah sirsak pada adonan roti
sebesar 50% dengan lama fermentasi yeast segar buah sirsak selama 72 jam dan T4 : penambahan
yeast segar buah sirsak pada adonan roti sebesar 50% dengan lama fermentasi yeast alami selama
96 jam.

Menurut Ko (2012) penambahan yeast segar kismis pada adonan roti 50% dari berat tepung terigu
yang dipakai. Masing-masing perlakuan dilakukan sebanyak 4 kali ulangan. Data yang diperoleh
dianalisis dengan ANOVA dan jika terdapat perbedaan maka dilakukan dengan Uji Wilayah Ganda
Duncan.

Hipotesis yang diuji dalam penelitian ini adalah adanya pengaruh lama fermentasi yeast segar buah
sirsak terhadap variabel yang ditetapkan yaitu : mutu kimia (pH, kadar alkohol, gas yang dihasilkan),
fisik (daya kembang adonan) dan mikrobiologis (viabilitas mikroba segar buah sirsak) hasil
fermentasi yeast segar buah sirsak.

H0 : Tidak ada pengaruh lama fermentasi yeast segar buah sirsak terhadap mutu kimia, fisik dan
mikrobiologis pada hasil fermentasi yeast segar buah sirsak buah sirsak.

H1 : Ada satu pengaruh lama fermentasi yeast segar buah sirsak terhadap mutu kimia, fisik dan
mikrobiologis pada hasil fermentasi yeast segar buah sirsak.

Secara statistik, hipotesis empirik di atas dapat dijabarkan ke dalam hipotesis statistik sebagai
berikut:

H0 :0= 1 = 2 = 3 = 4

H1 :0.......... 4

Kriteria pengujian analisis yang digunakan adalah sebagai berikut:

F hitung < F tabel artinya H0 diterima dan H1 ditolak

F hitung ≥ F tabel artinya H0 ditolak dan H1 diterima24

3.2.2. Variabel Penelitian

Variabel kualitas yeast segar buah sirsak yang diamati terdiri dari uji viabilitas mikroba, pengukuran
nilai pH, volume adonan roti yang dihasilkan, alkohol yang dihasilkan, daya kembang adonan roti,
produksi gas yang dihasilkan. Sub-sub berikut menyajikan analisis atau uji variabel tersebut :

a. Analisis Viabilitas Mikroba

Prinsip pengujian viabilitas mikroba dilakukan dengan metode TPC (Total Plate Count) yaitu dengan
menginkubasi sampel yeast pada media NA (Nutrient Agar) untuk mengetahui jumlah mikroba yang
bertahan hidup dan menghitung jumlah yeast tersebut. Hal tersebut sesuai dengan pendapat Fardiaz
(1992) yang menyatakan bahwa untuk menguji mikrobiologi dapat dilakukan dengan metode TPC.
Langkah awal dalam uji TPC adalah dengan sterilisasi alat terlebih dahulu dengan membungkus
cawan petri dan pengaduk dengan kertas pembungkus kemudian di oven dengan suhu 180 C selama
1 jam. Dan untuk gelas beker yang tidak tahan terhadap panas disterilisasikan dengan autoklaf
dengan suhu 121 C pada tekanan 2 atm selama 15 menit (Paliling et al., 2016). Langkah selanjutnya
adalah menyiapkan media NA yaitu dengan menimbang NA sebanyak 5 g dan dilarutkan dalam
aquades 250 ml yang dipanaskan, media yang sudah jadi di sterilisasikan dengan autoklaf pada suhu
121 C dengan tekanan 2 atm (Suhermawan, 2013).

Selanjutnya mengambil sampel sebanyak 1 ml sampel diencerkan dalam tabung reaksi yang berisi 9
ml aquades yang telah disterilisasikan (10^1) Pengenceran dilakukan hingga pengenceran (10^7)
cawan petri digoyang-goyangkan. Media dituangkan di masing-masing cawan dan ditunggu hingga
padat dan posisi cawan dibalik posisinya. Masukkan cawan kedalam inkubator pada suhu 35 C
selama 24-48 jam. Setelah di inkubasi media, amati jumlah koloni pada masing-masing cawan dan
dihitung. Kemudian sampel dihitung setelah diinkubasi selama 24 jam-48 jam dengan jumlah koloni
yang diterima yaitu 30-300 koloni. Nilai TPC dapat dihitung dengan rumus perhitungan koloni :

𝐽𝑢𝑚𝑙𝑎ℎ 𝑘𝑜𝑙𝑜𝑛𝑖 𝑝𝑒𝑟 𝑐𝑎𝑤𝑎𝑛 𝑥 1/ 𝐹𝑎𝑘𝑡𝑜𝑟 𝑝𝑒𝑛𝑔𝑒𝑛𝑐𝑒𝑟𝑎𝑛

b. Analisis Uji Nilai pH

Uji nilai pH dilakukan dengan menggunakan pH meter. Mengambil sampel yeast segar kismis
sebanyak 10 ml kemudian diencerkan dengan aquades sebanyak 90 ml. Sampel yang telah
diencerkan dihitung nilai pHnya dengan pH meter yang terlebih dahulu dikalibrasi menggunakan
larutan buffer pH 4 dan 7 agar didapatkan hasil pengujian yang akurat (Apriyanto et al, 1989).
Elektroda dibersihkan terlebih dahulu dengan aquades dan dikeringkan dengan menggunakan tissue,
kemudian dicelupkan pada larutan buffer dan dicelupkan ke sampel yang akan diuji tunggu beberapa
saat hingga pH meter berhenti pada angka pH yang stabil.

c. Analisis Kadar Alkohol

Menurut Anisa (2019) metode untuk menguji kadar alkohol menggunakan metode piknometer
dengan prinsip menghitung berat jenis alkohol. Piknometer yang digunakan untuk menghitung kadar
alkohol adalah piknometer dengan ukuran 10 ml. Pengujian melakukan destilasi terhadap sampel
sebanyak 100 ml dan dimasukkan kedalam labu kjeldhal dan dihubungan dengan alat destilator
kemudian dipanaskan dengan suhu 80 C. Hasil destilasi kemudian ditampung pada erlemeyer
sebanyak 80 ml. Hasil destilat dimasukkan kedalam piknometer yang telah diketahui berat
kosongnya. Sampel dimasukkan kedalam piknometer hingga penuh sebelum ditimbang terlebih
dahulu piknometer dibersihkan dengan menggunakan tissue untuk membersihkan sisa sampel yang
tersisa di bagian luar piknometer agar perhitungan kadar alkohol menjadi akurat. Melakukan
metode yang sama menggunakan aquades sebagai pembanding untuk mengetahui Berat Jenis (BJ)
kadar alkohol sampel.
Berat jenis alkohol dapat dihitung dengan rumus:

(berat piknometer + destilat) − berat piknometer kosong / (berat piknometer + aquades) − berat
piknometer kosong

Sampel yang telah diketahui berat jenisnya kemudian dikonversikan dalam tabel berat jenis alkohol
untuk menentukan kadar alkohol yang diperoleh.

3.2.4. Prosedur Penelitian

a. Pembuatan Yeast Segar (Starter)

Metode pembuatan yeast segar buah sirsak dilakukan sesuai dengan metode Ko (2012) yaitu
menyiapkan kismis yang telah matang. Sirsak dikupas dan dihilangkan biji hanya diambil daging
buahnya saja. Toples kaca yang akan digunakan disterilisasi terlebih dahulu dengan cara direbus
selama 5 menit untuk membunuh bakteri lain yang kemungkinan terdapat didalam toples kaca yang
akan digunakan untuk fermentasi sehingga tidak akan mengkontaminasi saat fermentasi. Kemudian
kismis dimasukkan kedalam toples kaca sebanyak 100 gr ditambahkan 250 ml aquades dan 30 gr
gula pasir dan kemudian di fermentasikan selama 0 jam, 24 jam, 48 jam, 72 jam dan 96 jam dan
dimasukkan kedalam box Styrofoam untuk menjaga suhunya stabil. Diagram alir proses pembuatan
yeast segar buah sirsak disajikan pada Lampiran 1.

3.2.5. Analisis Data

Data yang didapatkan dari uji kadar alkohol, uji pengukuran gas, uji daya kembang, uji viabilitas
mikroba dan uji pH diolah dengan menggunakan RAL (Rancangan Acak Lengkap) dengan tingkat
kepercayaan 95%. Data yang didapatkan kemudian dianalisis dengan mengunakan uji ANOVA
(Analysis Of Varian) menggunakan SPSS 16.0 Statistic Software pada taraf signifikansi 5%. Apabila
terjadi pengaruh nyata dilanjutkan dengan uji lanjut Wilayah Ganda Duncan untuk mengetahui
perbedaan perlakuannya, hipotesis penelitian apabila P value pada hasil analisis < sig 0,05.

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---------------------------------------------

Li, Z., Song, K., Li, H., Ma, R., & Cui, M. (2019). Effect of mixed Saccharomyces cerevisiae Y10 and
Torulaspora delbrueckii Y22 on dough fermentation for steamed bread making. International Journal
of Food Microbiology. doi:10.1016/j.ijfoodmicro.2019.05.009

Domesticated Saccharomyces cerevisiae, also known as baker's yeast, can produce high amounts of
gas and is widely used in dough fermentation for bread making because of its simplicity and short
processing time (De Vuyst et al., 2016; Jayaram et al., 2013; Rezaei et al., 2014). The use of selected
S.

cerevisiae strains helps control the dough fermentation process and reduces the risk of spoilage.
However, this method decreases the diversity of microflora and produces bread that lacks distinctive
characteristics, such as unique texture, taste and flavour, compared with sourdough (Keeratipibul et
al., 2013; Wang et al., 2018; Wu et al., 2012)

T. delbrueckii is also found on grape surface, and its strains show variation in their ability to ferment
and

assimilate carbon compounds (van Breda et al., 2013). T. delbrueckii has been previously assessed
for its possible contribution to dough fermentation and bread making and for its good gas yield and
satisfactory flavour as compared with non-Saccharomyces yeasts (Aslankoohi et al., 2016;
Hernandez-Lopez et al., 2003). The biotechnological interest in T. delbrueckii has recently increased
because of its high freezing and osmotic tolerance (Almeida and Pais, 1996; Hernandez-Lopez et al.,
2003; Pacheco et al., 2012)

S. cerevisiae plays a dominant role in the glycolysis of glucose and rapidly increases the consumption
of sugars

(Chen and Liu, 2016). In the current study, T. delbrueckii was less efficient than S. cerevisiae in
utilising sugars as a culture. The leavening ability of dough was largely dependent on carbohydrate
composition and yeast utilisation (Alves-Araújo et al., 2007; Zhou et al., 2017). Therefore, the carbon
source utilisation behaviour showed the potential of the two yeasts for use as co-culture in dough
fermentation

to increase the persistence of CO2 production and improve product quality

The net increase in succinic acid and acetic acid because S.

cerevisiae produces these acids (Jayaram et al., 2013). Spontaneously co-growing LAB produces a
mixture of acetic acid and lactic acid (Gamel et al., 2015).

Appropriate pH and amounts of lactic acid and acetic acid can improve the product volume and gas
retention capacity of the dough (Gobbetti, 1998; Rocha and Malcata, 2012). Biological acidification
greatly increases loaf specific volume relative to the chemical acidification (Clarke et al., 2002). On
the contrary, organic acids in the dough can increase the baker's yeast activity (increased CO2
release), which subsequently results in good dough development (Moroni et al., 2012). However,
extremely high acid concentration negatively affects the yeast growth and activity (Ua-Arak et al.,
2017).

Yeast metabolism is the main source of aromatic diversity in alcoholic beverages and bread (Birch et
al., 2013; De Vuyst et al., 2016). Thus, further studies must investigate synergism for aromatic
diversity production in culture.

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Pengukuran Brix
Indeks bias merupakan salah satu dari beberapa sifat optis yang penting dari medium suatu bahan.
Nilai indeks bias ini banyak diperlukan untuk menginterpretasi suatu jenis data spektroskopi. Indeks
bias dari suatu bahan atau larutan merupakan parameter karakteristik yang sangat penting dan
berkaitan erat dengan parameter-parameter lain seperti temperatur, waktu, pH, kadar etanol,
konsentrasi, jumlah mikroorganisme dan lain-lain yang sering dipakai dalam bidang optik, kimia,
industri pangan dan obat-obatan (Sutiah, 2008).

Indeks bias suatu larutan dapat diukur dengan menggunakan beberapa metode salah satunya
menggunakan spektrometer dan refraktometer. Alat refraktometer modern mempunyai metode
yang berbeda dalam pengukuran indeks bias, jangkauan pengukuran, tingkat akurasi, metode yang
digunakan untuk merekam pergeseran cahaya, sifat dari sumber cahaya, pembuatan perangkat
sampling dan pengukuran sel.

Indeks bias mutlak suatu medium adalah rasio dari kecepatan gelombang elektromagnetik dalam
ruang hampa dengan kecepatannya dalam media tersebut. Indeks bias relatif adalah rasio dari
kecepatan cahaya dalam suatu medium ke dalam medium yang berdekatan. Refraksi terjadi pada
semua jenis gelombang, tetapi pada umumnya terjadi pada gelombang cahaya. Indeks bias medium
memiliki panjang gelombang yang berbeda-beda. Suatu efek yang dikenal sebagai dispersi,
memungkinkan prisma memisahkan cahaya putih menjadi warna penyusunnya. Untuk warna
tertentu, indeks bias medium bergantung pada kerapatan medium, yang juga merupakan fungsi dari
konsentrasi. Nilai indeks bias refraktometer juga dikenal sebagai nilai °Brix. Nilai °Brix sendiri
merupakan ukuran total padatan terlarut dalam larutan yang berkorelasi erat dengan fraksi molar
komponen dan memiliki nilai konstan untuk suatu zat pada kondisi suhu dan tekanan standar. Brix
telah banyak digunakan untuk menentukan konsentrasi zat-zat seperti pada industri obat-obatan,
makanan, jus buah, formula diet, larutan nutrisi parenteral, dan industri bir serta wine (Chang et al,
2004).

Pengukuran menggunakan refraktometer konvensional cenderung rumit dan memakan waktu yang
lama sehingga dibutuhkan refraktometer simpel dan bersifat portabel yang dapat mengukur indeks
bias secara mudah, cepat, dan akurat. Hal ini lah yang mendasari penggunaan Hand Brix
Refraktometer. Hand Brix Refraktometer bekerja menggunakan prinsip pembiasan cahaya ketika
melalui suatu larutan. Ketika cahaya datang dari udara ke dalam larutan, maka kecepatannya akan
berkurang. Fenomena ini terlihat pada batang yang terlihat bengkok ketika dicelupkan ke dalam air.
Hand Brix Refraktometer memakai prinsip ini untuk menentukan jumlah zat terlarut dalam larutan
dengan melewatkan cahaya ke dalamnya. Sumber cahaya ditransmisikan oleh serta optik ke dalam
salah satu sisi prisma dan secara internal akan dipantulkan ke interface prisma dan sampel larutan.
Bagian cahaya ini akan dipantulkan kembali ke sisi yang berlawanan pada sudut tertentu yang
tergatung dari indeks bias larutannya (Pedrotti, 1993).

Metode analisis kuantitatif refraktometrik pada berbagai media cair berkembang lebih pesat dan
lebih luas menggantikan metode yang volumetri dan gravimetri yang lebih banyak memakan waktu
dan kurang akurat.
Gambar Hand Brix Refraktometer

Cara kalibrasi alat Hand Brix Refraktometer:

a). Letakkan satu atau dua tetes akuades diatas kaca prisma

b). Tutup penutup kaca prisma dengan perlahan

c). Pastikan akuades memenuhi permukaan kaca prisma

d). Pembacaan skala melalui lubang teropong, pastikan garis batas biru tepat pada skala 0°Brix

e). Jika garis batas biru tidak tepat pada skala 0°Brix, putar skrup pengatur skala hingga garis batas
biru tepat pada skala 0°Brix.

Cara penggunaan alat Hand Brix Refraktometer :

a). Alat dibersihkan terlebih dahulu dengan tisu ke arah bawah

b). Ditetesi dengan akuades atau larutan NaCl 5% pada bagian prisma dan day light plat

c). Dibersihkan dengan kertas tissue sisa akuades/NaCl yang tertinggal

d). Sampel cairan diteteskan pada prisma 1-3 tetes

e). Skala kemudian dilihat di tempat yang bercahaya dan dibaca parameter skalanya

f). Kaca dan prisma dibilas dengan aquades/NaCl 5% serta dikeringkan dengan tisu

g) Alat disimpan di tempat kering

Preparasi Sampel dan Pengukuran Brix (Wahyudi, 1997)

Sampel sebanyak 3-5 tetes dimasukkan dalam alat pengukur Brix (Hand brix refraktometer) tepatnya
pada celah antara prisma penutupnya yang kering dan basah. Diamati batas tajam antara garis
terang dan gelap tepat pada titik potong sumbunya. Dengan mengatur ketepatan batas tersebut
hingga jelas, maka dapat diketahui skala (berupa angka) dan tidak boleh terlihat garis pelangi
diantaranya.

Refraktometer tipe hand-held merupakan salah satu alat

yang dapat digunakan untuk menganalisis kadar sukrosa

pada bahan makanan. Refraktometer terdiri atas beberapa

bagian, yaitu kaca prisma, penutup kaca prisma, sekrup

pemutar skala, grip pegangan, dan lubang teropong (Atago

2000) (Gambar 1). Satuan skala pembacaan refraktometer yaitu

°Brix, yaitu satuan skala yang digunakan untuk pengukuran

kandungan padatan terlarut (Purwono 2002). Skala °Brix dari

refraktometer sama dengan berat gram sukrosa dari 100 g

larutan sukrosa. Jika yang diamati adalah daging buah, skala

ini menunjukkan berat gram sukrosa dari 100 g daging buah

Persiapan Alat

Refraktometer perlu dikalibrasi terlebih dahulu sebelum

digunakan untuk satu hari pengamatan. Jika terjadi perubahan suhu, alat ini perlu dikalibrasi
kembali. Cara mengkalibrasi refraktometer dimulai dengan membuka penutup kaca prisma,
kemudian di atas kaca prima diteteskan satu atau dua tetes akuades. Penutup kaca prisma lalu
ditutup lagi dengan perlahan dan dipastikan akuades memenuhi permukaan kaca prisma.
Refraktometer diarahkan pada cahaya terang, kemudian dilihat pembacaan skala melalui lubang
teropong. Jika skala kabur, lubang teropong diputar hingga pembacaan skala tampak jelas. Pastikan
garis batas biru tepat pada skala 0°Brix (% maks. sukrosa). Jika garis batas biru tidak tepat pada skala
0°Brix, sekrup pengatur skala diputar hingga garis batas biru tepat pada skala 0°Brix. Setelah kalibrasi
selesai, kaca prisma dibersihkan dengan menggunakan kertas tisu.

Pelaksanaan Pembacaan

Untuk membaca hasil analisis kandungan sukrosa buah pepaya dengan refraktometer, penutup kaca
prisma dibuka lalu di atasnya diletakkan satu atau dua tetes sari buah dari

masing-masing aksesi. Contoh sari buah dipastikan memenuhi permukaan kaca prisma, lalu penutup
kaca prisma ditutupkan kembali secara perlahan. Pembacaan skala dilakukan pada posisi garis batas
biru. Hasil pembacaan skala dicatat pada kertas pengamatan. Setelah pembacaan, kaca prisma
dibersikan dengan kertas tisu basah.

Tahapan kalibrasi alat refraktometer: (a) letakkan satu

atau dua tetes akuades di atas kaca prisma, (b) tutup

penutup kaca prisma dengan perlahan, (c) pastikan akuades memenuhi permukaan kaca prisma, (d)
pembacaan skala melalui lubang teropong, (e) pastikan garis batas biru tepat pada skala 0°Brix (%
maks sukrosa), dan (f) jika garis batas biru tidak tepat pada skala 0°Brix, putar sekrup pengatur skala
hingga garis batas biru tepat pada skala 0°Brix (Balitbu Tropika Solok, 2002)

--------------------------

Hocking M.B. (1985) Fermentation Processes. In: Modern Chemical Technology and Emission
Control. Springer, Berlin, Heidelberg. https://doi.org/10.1007/978-3-642-69773-9_14

Strictly speaking, fermentation is the process of anaerobic breakdown or fragmentation of organic

compounds by the metabolic processes of microorganisms. The specific definition includes micro-
biological processes carried out under anaerobic (fermentative, or in the absence of air), aerobic
(respiratory), and enzymatic (via extracts) conditions.

From probable early beginnings of this kind, yeasts have been used in the arts of wine, beer, and
spirits production, as well as for the in situ production of the carbon dioxide used for leavening of
bread and other bakery goods.

Baker's yeast, yeast from petroleum and petroleum products, and vinegar are all presently in large
scale

production using continuous culture methods [3,5,6]. Even in the highly market sensitive brewing [7]
and wine making [8, 9] industries, which have traditionally operated exclusively by batch
fermentation and are still dominated by this mode of operation, continuous mode fermentations
have now been proved to be feasible.

The requirements for producing a specific product via an industrial process using fermentation are,
first and foremost, a culture of a specific microorganism that produces the desired end product [10,
11]. This is usually the dominant product, such as during the anaerobic production of ethanol (ca. 95
% selective) from sugar by yeasts, in which case the process is termed homofermentative. On the
other hand fermentations which produce several products, such as the anaerobic production of
butanol (60-70 %), acetone (20-30 %), and ethanol (ca. 10 %) from sugars by Clostridium
acetobutylicum [12], may also be useful if all the products are recoverable and saleable. In this case
the process is termed heterofermentative.

*ca = roughly, approximately, more less (kira-kira, kurang lebih)

Raw materials appropriate for conversion must be economic for the end product desired, i.e. sugars
and starches for beverage alcohol, low cost starches or cellulosic substrates for fuel alcohol, etc.
Acceptable to good yields should be produced, based on the relative prices of substrate and end
product. The fermentation should proceed rapidly under conditions (pH, temperature, pressure,
nutrients, etc.) that can be relatively easily provided.
Cultures of the desired strains are maintained in dormant state and supplemented under scrupulous
conditions. Portions of the cultures can then be taken, and provided with ideal propagating
conditions to build up the numbers of organisms available to provide a 'starter' for inoculation to the
medium of a full scale fermenter. Yeasts, for example, in the presence of air ferment simple sugars
to carbon dioxide and water, yet under anaerobic conditions produce ethanol instead. It should be
noted that even anaerobic fermentations, such as are used for alcohol production, can be conducted
in open-topped fermenters when these are located inside a building.

Industrial Ethyl Alcohol

Before 1945, most of the supply of ethyl alcohol

for industrial solvent or feedstock uses was derived

from fermentation sources (Table 14.12). Since this time, the reliability and low cost of
petrochemical routes to the product caused a rapid replacement of fermentation sources by
synthetic routes in the U.S.A. The early petrochemical sources were based on the hydrolysis of ethyl
sulfate, but more lately this has been dominated by processes based on the direct gas phase
hydration of ethylene [71] (Equation 14.18-14.20).

CH2=CH2 + H2SO4 --> CH3CH2OSO3H (14.18)

CH3CH2OSO3H + H2O --> CH3CH2OH + H2SO4 (14.19)

CH2=CH2 + H2O --> CH3CH2OH (14.20)

Since 1975 however, increased costs and scarcity of the petroleum resource appear to have caused a
reversal of this trend and have renewed the interest by producers in fermentation routes to ethanol
[72]. Several recent announcements of new construction plans for large fermentation units based on
a variety of substrates strengthen the indirect evidence of the reversal of this trend [73-77], and at
least one distillery formerly used for whisky production has been converted to industrial alcohol
production [78].

Starches, and fruit, or sugarcane sugars can be, and are used for industrial alcohol production in a
similar manner to that used for the beverage products [79-81].

Fermentation temperatures are far less critical for spirits production than for beer, since the
important flavour elements are not affected by temperature in the same way. Since only a few
minutes at 50 ° C is enough to kill the yeast [6], however, sufficient cooling is applied to keep the
closed fermenter temperature below about 38°C to avoid this. The higher fermentation
temperatures used here speeds up the process significantly to complete the fermention in a period
of only about 3 days instead of the 6 or 7 days required for the refrigerated fermentation of beer.
Under these conditions, by the time 50 % of the sugar in the mash has been converted there is no
further yeast propagation. From this point on the remaining sugar to alcohol conversion is
accomplished by metabolism by the yeast population already present. The product of the mash
fermentation is a "beer" containing 7.5 to 8 % alcohol by volume. This name does not imply any
beverage qualities to the product at this stage. It is merely a reflection of the similarities of the
methods used to obtain it. Beverage spirits are obtained from this fermentation product by various
distillation, blending, and aging procedures.

Beverage Spirits

Spirits represent a class of alcoholic beverage which generally have an alcohol content of 20 % or
more, above the range achievable solely by fermentation. Thus the spirits classification includes
those beverages where distillation is necessary to raise the alcohol content above that achievable by
fermentation and where the distillate forms an essential and significant part of the final product.

An 'off' wine may also be converted to wine vinegar by acetification (Equation 14.15), a process that
is sufficiently profitable that at least one California winery has specialized in producing wine vinegar,
rather than wine [6].

Acetobacter acetii (14.15)

CH3CH2OH + O2 ---------------> CH3COOH + H20

Vinegar production, as mentioned earlier, represents one of the fermentation processes that is
practised both on a batch basis, literally 'in the barrel', and in continuous flow modes where the
acetifying wine is continuously recirculated over a bacterial support. With care and appropriate local
circumstances for byproduct sale and disposal, few of the byproducts and waste materials of a
winery operation need to be wasted.

Acid and glycerin formation, small but important components of both white and red wines, tend to
increase towards the end of the fermentation under the influence of the inhibiting ethanol (Equation
14.12).

yeast

2C6H12O6 + H2O --> CH3CH2OH + CH3COOH + 2CH2OHCHOHCH2OH +

2CO2 (14.12)

Principal Steps of Winemaking

Large numbers of natural yeasts (and other microorganisms) are found adhering to the fruit in the
field. These are produced by starting with an initial sugar content which is higher than can be
completely fermented by the yeast. [27].

The art of wine making, like brewing, was developed well before the beginnings of recorded history.
That these skills were acquired by experimentation following the accidental fermentation of stored
fruit or fruit juices by natural yeasts which were originally present on the surface of the fruit. It is
thought that viticulture originated in the lands south of the Caspian Sea since signs of grape
cultivation and winemaking have been discovered in Mesopotamia dating back 4,500 years. Certainly
winemaking and aging skills were well known to

the Greeks, some 2,500 years ago [38]

From a chemical standpoint winemaking is similar to brewing since the essential alcohol-forming
step still involves an anaerobic fermentation using yeast. But there are also some significant
differences. The key distinction is that winemaking starts with a fruit, usually grape, which already
contains sugar, the fermentation substrate. Thus many of the complicated preliminary steps
required in brewing to produce a sugar solution for fermentation from starch-based raw materials,
are unnecessary for winemaking. This greatly simplifies the early steps of the winemaking process.

A number of varieties of grapes form the dominant winemaking raw material but many fruits other
than the grape, such as apples, loganberries, raspberries, strawberries, etc. are also used. Apart from
the vested incentive to produce an appealing beverage product, there is also an economic
valueadded incentive to winemaking from grapes on a large scale. To illustrate this, using Canadian
mid1973 prices, grapes sold wholesale at some

190 $/tonne and retailed fresh at something like 660 $/tonne [39]. To contrast with these figures,
the roughly 600 to 800 L of wine obtainable from the tonne of grapes (ca. 160 Imp. gal/ton grapes)
was worth some $1,600, a coarse measure of the profitability, or the value-added incentive.
Consequently, half or more of the grapes grown go into winemaking. In Canada, in the mid 1970's,
the

proportion going into winemaking was over 75 %.

Brewing of Beer

Certainly beers, beverages produced from a fermented brew of cereal(s) and hops, have been
produced at least since the dawn of recorded history and probably well before that [14-17]. The
manufacture of beer is an example of an anaerobic fermentation process which uses varieties of
yeast to produce a beverage of low alcohol content from basically a solution of simple sugars in
water.
The principal steps of beer production in a modern, large scale brewery [20]. Reprinted courtesy of
Southam Business Publications, Ltd.

Heat evolution of 74.5 to 93.3kJ, depending on the

source of combustion data used [25, 26], is evolved

for each mole of glucose fermented to ethanol and

carbon dioxide (Equation 14.8).

zymase

C6H12O6 ----> 2CH3CH2OH + 2CO2 (14.8)

(yeast)

Delta H = -74.5 to - 93.3 kJ (-17.8 to - 22.3 kcal)

The heat evolution on maltose fermentation, which involves a preliminary mildly exothermic
hydrolytic step to glucose before alcohol formation (Equation 14.9), can be approximated by
multiplying the figure taken for glucose by two. A closer estimate may be obtained by adding the
further 15.9kJ/mole required for maltose hydrolysis [26] to this number.

maltase

C12H22O11 + H2O ----> 2C6H1206 (14.9)

Delta H = -15.9 kJ (- 3.8 kcal)

Fermentation temperatures of 15 to 18°C are desirable for beer production. Above this range some
flavour elements are lost and head (froth) formation of the finished beer is poor. Therefore, brewers
normally use refrigeration to maintain fermentation temperatures in this optimum range.

Yeasts used in brewing (and for winemaking and spirits production) are homofermentative, that is,
under anaerobic conditions they are about 95 % selective for ethanol as the end product of glucose
metabolism [27].

Using these equations it is possible to calculate the approximate alcohol concentration to be

expected on

fermentation of any given initial concentration of

fermentable sugars. During the fermentation step,

virtually all of the fermentable sugars present, comprising about 2/3 of the original carbohydrate

mashed, is converted to ethanol and carbon dioxide

(Table 14.3).--> Belum di salin di excel

Table 14.3 Breakdown of the principal carbohydrate

constituents of a normal wort used for making beer (Data selected from the more detailed listing
given in [7])

After fermentation the brew is separated from the yeast by centrifuging, or by decantation through
an outlet raised slightly above the bottom of the fermenter, which leaves nearly all of the yeast
behind as a 'heel' in the fermenter. Each fermentation produces enough yeast for the pitching
(starting) of five further brews of equivalent size [18]. After careful checking for strain purity and
freedom from mutations the middle portions of this yeast will be reused, sometimes for as long as
three

months or 8 to 12 fermentations, before it is arbitrarily discarded and a starter freshly prepared

from pure culture stock used to re-initiate the whole process. The decanted fermented brew is
filtered and the opalescent product at this stage is referred to as 'green' beer.

Lager beers, which would follow the proportions tabulated fairly closely, have two distinctions. First
they are produced by a bottom-fermenting strain of yeast variety of Saccharomyces uvarum or a
Saccharomyces carlsbergensis -named after its discovery and isolation by E.C. Hansen at the
Carlsberg Institute, Copenhagen, in about 1870- [18]. Bottom fermentating yeasts settle out or sink
in the green beer on completion of fermentation.

Ale, the basic difference being that it is produced by

a top-fermenting yeast, Saccharomyces cerevisiae, or a related strain. Not only is the yeast skimmed
from the top of the green beer, on completion of fermentation, but fermentation is carried out at 15
to 20 a C, slightly higher than required for lagers, and therefore requiring somewhat shorter times
[28, 30].

The perishability of beer is attributable to its low

alcohol content, which is inadequate to prevent attack by micro-organisms, and to the unstable
nature of many of its constituents. Thus, bottled and draught beers are at their best immediately
after packaging. Optimum keeping qualities are obtained by ensuring minimum oxygen content [31].

[3] J.E. Bailey and D.F. Ollis, Biochemical Engineering Fundamentals, McGraw-Hill, New York, 1977
(Bibliography)
[3]-Reference

L.M. Miall, Royal Inst. of Chern. Reviews 3 (2), 135, Oct. 1970

[5] Industrial Aspects of Biochemistry, Volume 30, Parts I and II, B. Spencer, editor, North-Holland
Publishing Co., Amsterdam, 1974 (Bibliography)

[5]-Reference

Fermentation Unit Runs Nonstop for 75 Days, Chern. Eng. News 58(22), 27, June 2, 1980

[6] G.L. Solomons, Materials and Methods in Fermentation, Academic Press, New York, 1969
(Bibliography)

[6]-Reference

M.A. Amerine, H.W. Berg, and W.V. Cruess, The Technology of Wine Making, 2nd edition, Avi
Publishing Co., Westport, Conn., 1967

[7] Chemistry of Winemaking, ACS Advances in Chemistry Series No. 137, American Chemical
Society, Washington, 1974 (Bibliography)

[7]-Reference

Kirk-Othmer Encyclopedia of Chemical Technology, 3rd edition, John Wiley and Sons, New York,
1978, volume 3, page 692

[8] Wine Production Technology in the United States, ACS Symposium Series No. 145, American
Chemical Society, Washington, 1981 (Bibliography)

[8]-Reference

A. Weisenberger, Food Eng. 50(6), 136, June 1978

[9 & 10] Applied Biochemistry and Bioengineering, L.B.

Wingard, Jr., E. Katzir, and L. Goldstein, editors,

Academic Press, New York, ca. 1978

Volume 1, Immobilized Enzyme Principles, 1976

Volume 2, Enzyme Technology, 1979

(Bibliography)

[9]-Reference

G. Anton and A.M. Vicente, Food Eng. 51 (2), 76, Feb. 1979

[10]-Reference

Riegel's Handbook of Industrial Chemistry, 7th edition, J.A. Kent, editor, Van Nostrand/Reinhold,
New York, 1974, page 156.

[11, 12] Advances in Biochemical Engineering, T.K. Ghose, A. Fiechter, and N. Blakebrough, editors,
Springer-Verlag, Berlin

Volume 10, Immobilized Enzymes I, 1978

Volume 11, Microbiology, Theory and Application, 1979

(Bibliography)

[11]-Reference

R.N. Shreve and J.A. Brink, Jr., Chemical Process

Industries, 4th edition, McGraw-Hill, New York, 1977, page 525

[12]-Reference

W.N. McCutchan and R.J. Hickey in Industrial Fermentations, L.A. Underkofler and R.J. Hickey,
editors, Chemical Publishing Co., New York, 1954, volume 1, page 347.

[14] Immobilized Enzymes in Food and Microbial Processes, A.C. Olson and C.L. Cooney, editors,
Plenum Press, New York, 1974 (Bibliography)

[14]-Reference

C. Dagleish, La Recherche 11 (110), 434, April 1980

[15] P.A. Hahn, Chemicals from Fermentation, Doubleday, Garden City, New York, 1968
(Bibliography)

[15]-Reference

M.E. Chavez, Liquor: The Servant of Man, Little, Brown and Co., Boston, 1965. Cited by F.K.E. Imrie,
Chern. Ind. (London) 584, July 17, 1976

[16] C.S. Pederson, Microbiology of Food Fermentations, 2nd edition, Avi Publishing Co., Westport,
Connecticut, ca. 1979(Bibliography)

[16]-Reference

H.S. Corran, A History of Brewing, David and Charles Publishing Co., Newton Abbott, U.K., 1975

[17] F.W. Salem, Beer, Its History and Economic Value as a National Beverage, Arno Press, New York,
1972, reprint of 1880 edition.

[18] A.H. Rose, Scientific American 245 (3), 126, Sept.

1981

[20] T. Lorn, Can. Chern. Proc. 58(12),14, Dec. 1974

[25] M.S. Kharasch, Bureau of Standards J. of Research

2,359 (1929), cited by Handbook of Chemistry and Physics, 39th edition, C.D. Hodgman, editor,
Chemical Rubber Pub!. Co., Cleveland, Ohio, 1957, and by Lange's Handbook of Chemistry, 10th
edition, N.A. Lange, editor, McGraw-Hill,

New York, 1969

[26] J.F. Thorpe and M.A. Whiteley, Thorpe's Dictionary of Applied Chemistry, 4th edition, Longman's
Green and Company, London, 1941, volume 5, page 8

[27] M.A. Amerine, Scientific American 211 (2), 46, Aug. 1964

[28] R.I. Tenney, The Brewing Industry, in Industrial

Fermentations, L.A. Underkofler and R.J. Hickey, Chemical Publishing Co. Inc., New York, 1954,
volume 1, page 172

[30] F.H. Thorp and W.K. Lewis, Outlines of Industrial

Chemistry, The MacMillan Company, New York, 1917, page 444

[31] Z. Valyi and G.E.A. Van Gheluwe, Tech. Quart. Master Brewers' Assoc. of America 3, 184 (1974)

[38] H.W. Allen, A History of Wine, Horizon Press, New York, 1961

[39] L. Morisset-Blais, Can. Chern. Proc. 57(12), 31,

Dec. 1973

[71] F.A. Lowenheim and M.K. Moran, Faith, Keyes, and Clark's Industrial Chemicals, 4th edition,
Wiley-Interscience, New York, 1975

[72] U.S. Output of Synthetic Ethanol to Cease, Chern. Eng. News 61 (18), 18, May 2, 1983

[73] Ethanol Fuel from Wood, Can. Chern. Proc. 66 (1), 8, Feb. 19, 1982

[74] USDA Process Converts Xylose to Ethanol, Chern. Eng. News 59 (25), 54, June 22, 1981

[75] B. Orchard, Can. Chern. Proc. 64 (8),55, Nov. 26,

1980

[76] Ethanol for Gasoline Trucked to California, Chern.

Eng. News 57 (26), 10, June 25, 1979

[77] Ethanol Joint Venture Formed, Chern. Eng. News 58 (29), 16, July 21, 1980

[78] National Distillers to Convert Plant for Gasohol,

Chern. Eng. News 58 (19), 8, May 12, 1980

[79] W.L. Faith, D.B. Keyes, and R.L. Clark, Industrial Chemicals, 3rd edition, John Wiley and Sons,
New York, 1965

[80] J.G. da Silva, G.E. Serra, J.R. Moreira, J.C. Concalves, andJ. Goldemberg, Science 201 , 903, Sept.
8, 1978

[81] W.H. Long, Chern. Eng. News 59 (36), 7, Sept. 7,

1981

------------------------------------------

Konsentrasi gula yang terlalu tinggi akan menghambat aktivitas khamir dan waktu fermentasi yang
dibutuhkan menjadi lebih lama dengan konsekuensi tidak semua gula dapat difermentasi.
Sebaliknya, jika konsentrasi gula terlalu rendah akan mengakibatkan biaya produksi lebih tinggi (Kirk,
1963).

Menurut Manshur (1998) pembuatan etanol dapat dilakukan dengan dua cara yaitu :

1. Cara Sintesis

Pada cara sintesis dilakukan reaksi kimia untuk mengubah bahan baku menjadi alkohol. Misalnya
dengan reaksi hidrasi etilena yang merupakan hasil samping pada proses penyulingan minyak bumi.
Reaksi yang terjadi dapat dituliskan sebagai berikut : C2H4 + H2O -> C2H5OH

2. Cara Fermentasi

Cara ini dilakukan dengan menggunakan aktivitas mikroba. Pada proses fermentasi akan dihasilkan
bioetanol. Bioetanol adalah etanol yang dihasilkan dari biomassa atau bahan baku alami melalui
proses fermentasi. Produksi bioetanol dengan bahan baku tumbuhan berupa sereal, umbi, buah,
ataupun sayuran yang banyak mengandung pati atau karbohidrat akan dilakukan proses konversi
karbohidrat atau pati menjadi gula sederhana (glukosa) yang larut air dengan penambahan air dan
enzim. Selanjutnya gula sederhana yang terbentuk akan dilanjutkan proses peragian untuk
mendapatkan fermentasi alkohol oleh yeast yang mana gula sederhana tersebut akan di fermentasi
sehingga menghasilkan etanol.

Fermentasi Alkohol
Fermentasi adalah proses produksi energi dalam sel melalui respirasi anaerobik. Definisi lebih jelas
terkait fermentasi itu sendiri adalah sebuah proses respirasi dalam lingkungan anaerobik dengan
tanpa akseptor elektron eksternal (Winarno & Fardiaz, 1994). Fermentasi merupakan proses yang
relatif murah yang pada hakikatnya telah lama dilakukan oleh nenek moyang kita secara tradisional
dengan produk-produknya yang sudah biasa dikonsumsi oleh manusia hingga hari ini (Nurhayani,
2000).

Proses fermentasi tergantung pada banyak sedikitnya jumlah (viabilitas) dan kemampuan fermentasi
khamir yang digunakan. Semakin banyak jumlah khamir yang terlibat maka memiliki potensi untuk
menghasilkan kadar etanol yang tinggi (Tarigan, 1988). Semakin lama fermentasi, maka asam yang
dihasilkan akan semakin banyak pula. Proses terjadinya penurunan pH dapat terjadi di awal
fermentasi diakibatkan terbentuknya asam-asam organik selama proses fermentasi telah
berlangsung sejak awal. Asam-asam organik yang terbentuk seperti asam asetat, asam piruvat, dan
asam laktat mengakibatkan penurunan pH (Yuliani, 2003).

Khamir yang tumbuh dalam keadaan anaerobik akan cenderung melakukan proses fermentasi
alkohol yang mana substrat karbohidrat atau gula sederhana akan di gunakan untuk menghasilkan
etanol sebagai hasil samping sesuai jalur glikolisis Buckle (1987) :

Gula->Fosfogliseroldehid->Asam Piruvat

Anaerobik : Asam Piruvat->Asam Laktat->Alkohol->Etanol->Ester->Asam Asetat->Keton

Temperatur optimal untuk khamir berkisar antara 25-30°C. Beberapa jenis khamir dapat hidup pada
suhu 0-10°C, namun kemampuan fermentasinya lambat (Hidayat dan Suhartini, 2006).

Taksonomi khamir Saccharomyces spp. menurut Kavanagh (2005) adalah sebagai berikut:

Kingdom : Fungi

Division : Ascomycota

Class : Ascomycetes / Saccharomycetes

Ordo : Saccharomycetales

Familia : Saccharomycetaceae

Genus : Saccharomyces

Species : Saccharomyces cerevisiae

Khamir S. cerevisiae lebih banyak digunakan untuk memproduksi alkohol secara komersial
dibandingkan dengan bakteri. Hal ini disebabkan karena khamir dapat memproduksi alkohol dalam
jumlah besar. Namun S.cerevisiae sendiri umumnya tidak tahan pada etanol dengan konsentrasi
yang tinggi sehingga menyebabkan produktivitasnya menjadi rendah dan bahkan lethal
(Gunasekaran dan Raj, 1999).

Pada awalnya gula yang disakarida akan di pecahkan menjadi monosakarida atau gula sederhana
terlebih dahulu. Proses penguraian sukrosa dilakukan dengan bantuan enzim sukrase (invertase).
Sukrosa yang dipecahkan menghasilkan glukosa dan fruktosa (Wirahadikusumah, 1985). Skema
Embden Meyerhoff-Parnas Pathway sebagai berikut :

Inokulum adalah mikroorganisme yang diinokulasikan ke dalam sebuah medium, dimana

mikroorganisme tersebut masih dalam keadaan hidup atau masih berada pada fase pertumbuhan
yang sehat. Tujuan utama pembuatan inokulum adalah untuk starter dalam proses fermentasi.
Inokulum sebaiknya diinokulasikan ke dalam medium fermentasi pada saat sel aktif melakukan
metabolisme. Inokulasi adalah proses atau tahap kegiatan pemindahan mikroorganisme dari sumber
asalnya ke sebuah medium yang baru dan telah disediakan sebelumnya. Pembuatan inokulum pada
penelitian ini meliputi inokulum kerja dan inokulum induk. Inokulum kerja digunakan untuk
fermentasi yang mana dibuat dengan cara aseptik yaitu terbebas dari potensi pengaruh kontaminan
mikroorganisme lainnya.

Fermentasi spontan dilakukan dengan cara merendam bahan dalam air pada selang waktu tertentu
dengan memanfaatkan mikroba dari lingkungan. Selama perendaman tersebut terjadi perubahan
sifat yang disebabkan adanya aktivitas mikroorganisme antara lain adalah golongan ragi (yeast) dan
bakteri asam laktat (Hounhouigan et al, 1994). Sementara itu perbedaannya terhadap fermentasi
tidak spontan adalah dengan perlakuan penambahan starter mikroba spesifik yang akan digunakan
selama fermentasi ke dalam bahan (Johansson et al, 1995)

Suhu yang diperlukan untuk fermentasi alkohol adalah 20 - 30°C, kadang-kadang mencapai 35°C
pada akhir fermentasi (Wanto dan Soebagyo, 1980). Menurut Prescott dan Dunn (1981), suhu
optimal untuk fermentasi alkohol adalah 25 - 35°C. Kenaikan suhu akan menurunkan ketahanan
khamir terhadap alkohol yang dihasilkan dan akan meningkatkan pembentukan asam asetat yang
bersifat racun (Prescott dan Dunn, 1981). Sedangkan Frazier dan Westhoff (1978) menyatakan
bahwa suhu optimal pertumbuhan khamir pada fermentasi antara 25 - 30°C.

Hal ini disebabkan karena mikroba masih dalam fase adaptasi (fase lag) dimana sel masih
beradaptasi dengan kondisi lingkungannya. Pada fase ini mikroba merombak substrat menjadi nutrisi
untuk pertumbuhannya. Pada jam berikutnya yaitu memasuki jam ke-24 sampi jam ke-48 terlihat
adanya

percepatan pertambahan sel mikroba. Hal ini menandakan bahwa telah memasuki fase
pertumbuhan eksponensial (fase log). Kavanagh (2005) menyebutkan bahwa pada fase ini
Saccharomyces cerevisiae bereproduksi dengan membentuk tunas. Setelah jam ke-48, sel khamir
memasuki fase kematian yaitu ditandai

dengan jumlahnya yang mulai menurun, hal ini karena metabolit primer (bioetanol) yang dihasilkan
bersifat racun bagi khamir.

Dalam gambar 2 (a), fase stationer kurang terlihat karena pengamatan yang dilakukan pada periode
waktu yang terlalu lama (per 24 jam) sehingga fase stationer kurang terlihat. Akan tetapi fase
stationer tersebut dapat diperkirakan ada pada waktu sebelum dan sesudah 48 jam, karena pada
fase waktu 24-48 jam khamir mengalami fase eksponensial sedangkan pada fase waktu 48-72 jam
khamir mengalami fase kematian. Sehingga, berdasarkan estimasi tersebut, hasil pengamatan dapat
dikatakan serupa dengan hasil pada literatur gambar 2(b). Selain itu berdasarkan hasil pengamatan
dapat diketahui bahwa waktu kerja optimal khamir adalah pada jam ke-24 sampai jam ke-72. Setelah
jam ke-72 dapat disimpulkan bahwa proses fermentasi telah selesai dilakukan karena khamir telah
mati, sehingga apabila prose fermentasi dilanjutkan tidak akan berjalan efektif.

Perubahan pH disebabkan oleh adanya asam-asam organik seperti asam laktat, asetat dan piruvat
yang terbentuk selama proses fermentasi (Said, 1987). Menurut Reed dan Peppler (1973), asam-
asam yang terbentuk seperti asam asetat, asam piruvat, dan asam laktat dapat menurunkan pH,
sedangkan asam-asam lainnya seperti asam butirat dan asam lemak lainnya hanya sedikit
berpengaruh dalam penurunan pH cairan. Derajat keasaman akan mempengaruhi kecepatan
fermentasi, pH yang optimum untuk pertumbuhan khamir adalah 4-4,5 (Budiyanto, 2003).
Berdasarkan hasil pengamatan pH tersebut, dapat pula diketahui bahwa setelah 72 jam fermentasi
mulai berjalan kurang efektif yang disebabkan oleh tingkat keasaman larutan fermentasi yang
semakin menurun. Prescott dan Dunn (1981) menyatakan, pH pertumbuhan khamir yang baik antara
3.0 - 6.0. Perubahan pH dapat mempengaruhi pembentukan hasil samping. Pengaruh pH terhadap
pertumbuhan khamir juga tergantung pada

konsentrasi gula. Frazier dan Westhoff (1978), menyatakan bahwa pH akan mempengaruhi
kecepatan fermentasi, pH optimal untuk pertumbuhan khamir adalah 4.0 - 4.5. Oleh karena itu,
indikator pH/tingkat keasaman dapat dijadikan acuan untuk menentukan waktu optimal dan
berakhirnya proses fermentasi.

Ragi adalah suatu inokulum atau starter untuk melakukan fermentasi. Jenis ragi yang banyak
dimanfaatkan adalah Saccharomyces cereviciae sebagai mikroorganisme yang bertanggungjawab
terhadap proses fermentasi alkohol karena memiliki kemampuan produksi alkohol yang tinggi,
tahan terhadap kadar alkohol tinggi, tahan terhadap kadar gula yang tinggi dan tetap aktif
melakukan aktivitasnya pada suhu 4-32°C (Nurhayani, 2000).

Berdasarkan sumber mikroorganisme, proses fermentasi dibagi menjadi 2 yaitu :

1. Fermentasi Spontan, adalah fermentasi bahan pangan dimana dalam pembuatannya tidak
ditambahkan mikroorganisme baik dalam bentuk starter ataupun ragi, melainkan memanfaatkan
mikroorganisme yang berperan aktif dalam proses fermentasi berkembang baik secara spontan
karena lingkungan hidupnya dibuat sesuai untuk pertumbuhannya.

2. Fermentasi Tidak Spontan adalah fermentasi yang terjadi dalam bahan pangan yang dalam
pembuatannya ditambahkan mikroorganisme dalam bentuk starter atau ragi, dimana
mikroorganisme tersebut akan tumbuh dan berkembangbiak secara aktif merubah bahan yang
difermentasi menjadi produk yang diinginkan, contohnya pada pembuatan tempe dan oncom.

Kurva Pertumbuhan Mikroorganisme (Zahara, 2011)

Mikroorganisme mengalami beberapa fase pertumbuhan diantaranya adalah fase lag, fase
eksponensial, fase stasioner dan fase kematian.

1). Fase lag adalah kondisi dimana mikroorganisme baru saja diinokulasikan atau dibiakkan dalam
medium. Pada fase ini, mikroorganisme belum melakukan pembelahan, namun sudah terjadi
peningkatan massa volume, sintesis enzim, protein, dan peningkatan aktivita metabolik. Pada fase
tersebut bakteri lebih banyak melakukan adaptasi dengan lingkungan.

2). Fase eksponensial (Fase Log) adalah fase dimana mikroorganisme melakukan pembelahan secara
biner dengan jumlah kelipatan (eksponensial) secara logaritmik. Pada fase ini, terjadi lonjakkan
peningkatan jumlah biomassa sel, sehingga bisa diketahui seberapa besar terjadi pertumbuhan
secara optimal dan tingkatan produktivitas biomassa sel.

3). Fase stasioner adalah fase dimana bakteri sudah tidak melakukan pembelahan lagi. Ada 3
penyebab yang menyebabkan fase tersebut, diantaranya adalah ketidaktersediaan nutrient,
penumpukan metabolit penghambat dan produk akhir, serta kekurangan ruang gerak pada fase
stasioner yang disebut sebagai istilah "Lack of Biological Space".

4). Fase kematian (Fase Decline) adalah keterlanjutan dari fase stasioner dimana akan terjadi
pengurangan jumlah sel bakteri (biomassa) yang hidup. Fase kematian ditandai dengan jumlah sel
yang mati lebih banyak daripada sel yang hidup karena nutrien semakin menurun (bahkan habis)
sehingga energi cadangan di dalam sel juga habis. Selain itu akibat adanya beberapa kondisi stres
terhadap perubahan lingkungan yang dialami juga mempengaruhi kematian mikroorganisme.

Analisis Etanol

1. Uji Kualitatif Etanol

Uji kualitatif etanol digunakan untuk mengetahui ada tidaknya kandungan etanol di dalam suatu zat.
Uji kualitatif etanol perlu diterapkan pada penetapan kadar etanol metode bobot jenis karena untuk
mengetahui kapan destilasi harus dihentikan, hal ini penting untuk mengetahui bahwa etanol yang
didestilasi sudah benar-benar habis dan mencegah bercampurnya destilat dengan air.

a. Prinsip : Terbentuknya warna dan timbulnya bau

b. Cara : Etanol + Asam salisilat + Asam sulfat pekat Timbul bau harum dari etil salisilat

Etanol + Asam sulfanilat HCl + NaNO2 + NaOH Terbentuk warna

merah frambus (Departemen Kesehatan Republik Indonesia, 1989).

2. Uji Kuantitatif Etanol

Metode yang digunakan untuk menetapkan kadar etanol antara lain metode bobot jenis yang
merupakan metode konvensional dan kromatografi gas yang merupakan metode instrumental.

a. Metode Bobot Jenis

Definisi : Suatu metode penetapan etanol dengan perbandingan massa dari suatu zat terhadap
massa sejumlah volume air yang sama pada temperatur yang tertentu (Mardoni, dkk. 2006).

Prinsip : Sampel didestilasi kemudian destilat yang diperoleh ditetapkan bobot jenisnya. Dari bobot
jenis destilat maka dapat ditetapkan kadar etanolnya dengan menggunakan daftar bobot jenis (T. M,
Endang, dkk. 2005).

b. Metode Kromatografi Gas

Definisi : Suatu metode penetapan etanol dengan teknik kromatografi yang analisisnya berdasarkan
pada teknik pemisahan campuran atas dasar perbedaan distribusi zat-zat tersebut diantara fase
diam (cairan) dan fase gerak (gas), yang bisa digunakan untuk memisahkan senyawa organik yang

mudah menguap.

Prinsip : Fase diam dimasukkan kedalam kolom kemudian sampel

disuntikkan kedalam kolom. Senyawa akan terpisah dari campuran diantara fase gerak dan fase diam
sehingga zat tidak bercampur karena perbedaan kelarutan, dimana kelarutan dalam satu pelarut
lebih mudah dibanding dengan pelarut lainnya. Hasil akan keluar berupa puncak-puncak
(kromatogram). Setiap puncak mewakili satu senyawa. Kadar etanol ditetapkan dengan mengukur
waktu retensi dan membandingkan luas puncak sampel dengan luas puncak baku (Takeuchi, 2009).

Dasar-dasar Kromatografi

Kromatografi merupakan cara pemisahan yang mendasarkan partisi cuplikan antara fasa bergerak
dan fasa diam. Fasa bergerak dapat berupa gas atau cairan dan fasa diam dapat berupa cairan atau
padatan. Fase diam disini dapat berupa suatu zat padat yang ditempatkan dalam suatu kolo, atau
dapat juga berupa cairan terserap (teradsopsi) berupa lapisan yang tipis pada butir-butir halus pada
suatu zat padat pendukung (solid support material) yang ditempatkan di dalam kolom. Fase
geraknya dapat berupa gas (gas pembawa) atau cairan.

Campuran yang akan dipisahkan komponen-komponennya, dimasukkan kedalam kolom yang

mengandung fase diam. Dengan bantuan fase gerak, komponen-komponen campuran itu kemudian
dibawa bergerak melalui fase diam di dalam kolom. Perbedaan ataraksi atau afinitas atara
komponen-komponen campuran itu dengan kedua fase, menyebabkann komponen-komponen itu
bergerak dengan kecepatan berbeda melalui kolom.

Kelebihan dan Kekurangan Kromatografi Gas

1. Kelebihan

a. Waktu analisis yang singkat dan ketajaman pemisahan yang tinggi

b. Dapat menggunakan kolom lebih panjang untuk menghasilkan efisiensi pemisahan yang tinggi

c. Gas mempunyai vikositas yang rendah

d. Kesetimbangan partisi antara gas dan cairan berlangsung cepat sehingga analisis relatif cepat dan
sensitifitasnya tinggi

e. Pemakaian fase cair memungkinkan kita memilih dari sejumlah fase diam yang sangat beragam
yang akan memisahkan hampir segala macam campuran.
2. Kekurangan

a. Teknik Kromatografi gas terbatas untuk zat yang mudah menguap

b. Kromatografi gas tidak mudah dipakai untuk memisahkan campuran dalam jumlah besar.
Pemisahan pada tingkat mg mudah dilakukan, pemisahan pada tingkat gram mungkin dilakukan,
tetapi pemisahan dalam tingkat pon atau ton sukar dilakukan kecuali jika ada metode lain.

c. Fase gas dibandingkan sebagian besar fase cair tidak bersifat reaktif terhadap fase diam dan zat
terlarut.

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