APPROVAL SHEET

The complete report of Biochemistry Experiment with title is

“Fermentation“ was made by :
name : Wiwiana
ID : 1513441007
class : ICP Chemistry Education
group : IV (Four)
It has been checked and consulted by assistant and assistant coordinator, this
report has been accepted.

Makassar, November 2017

Assistant Coordinator Assistant

Muhammad Iqbal Hidayat Sriyanti, S. Pd

ID : 1313441008

Known by,
Responsibility Lecturer

Dr. Halimah Husain, M. Si

ID. 19641020 199003 1 002
A. TITLE OF EXPERIMENT
Fermentation
B. AIM OF EXPERIMENT
To study about ability fermented amilum, glucose, fructose, manos,
galactose by some pure inoculum bread yeast(Saccharomyces cerevisiae), tapai
yeast and pure inokulum Rhizopus oligosporus.
C. REVIEW LITERATURE
The carbohydrates are a group of naturally occurring carbonyl compounds
(aldehydes r ketones) that also contain several hydroxyl groups. The
carbohydrates include single sugars (monosaccharides) and their polymers, the
oligosaccharides and polysaccharides. Polymeric carbohydrates–above all starch,
as well as some disaccharides–are important (but not essential) components of
food. The form in which carbohydrates are distributed by the blood of vertebrates
is glucose (“blood sugar”). This is taken up by the cells and either broken down to
obtain energy (glycolysis) or converted into other metabolites.The
glycogenmolecules are covalently bound to a protein, glycogenin. Polysaccharides
are used by many organisms as building materials (Koolman, 2005 : 34).
The original penicillins isolated directly from mold fermentations were
mixtures of compounds having different side chains. The addition of phenylacetic
acid to the fermentation medium improved the yield of penicillin and ensured that
the product was substantially a single compound known as penicillin G or
benzylpenicillinThe main advantage of this change was an improvement in the
stability of the penicillin towards acid. These early penicillins, produced directly
by fermentation, were intensely active against Grampositive infections and gave
excellent results in streptococcal and staphylococcal infections and in
pneumonia (Frankin, 2005 : 33)
Over half of all biopharmacuticals thus far approved are produced in
recombinant E. Coli or S. cerevisiae. Industrial-scale bacterial and yeast
fermentation systems share many common features, an overview of which is
provided below. Microbial cell fermentation has a long history of use in the
production of various biological products of commercial significance A
generalized microbial fermenter design is presented in The impeller, driven by an
external motor, serves to ensure even distribution of nutrients and cells in the
tank. Various ports are also present through which probes are inserted which
monitor pH, temperature and sometimes the concentration of a critical metabolite
(Walsh, 2003 : 129).
Probiotics have received much attention on their proclaimed health
benefits which include improvement in lactose intolerance, increase in natural
resistance to infectious disease in gastrointestinal tract, suppression of
cancer, reduction in serum cholesterol level, and improved digestion. In
addition, there has been considerable interest in the effect of probiotics on
human lipid metabolism, and numerous studies have focused on the
potential hypocholesterolemic activity of probiotics in human. Furthermore,
one of the future challenges will be to unravel the physiological impacts of
bile salt hydrolase activity on the enzymeproducing bacterial and mammalian
cells (Kumar, 2012).
Youghurt is a special fermented milk product which have a texture of soft
and thicker compare to that of its raw material, the milk. The mild acid with
pleasant fresh taste supported characteristics of yoghurt as an extraordinary
fermented milk product. Bioactive peptides produced from hydrolysis of casein in
milk generated by L. Helveticus have been reviewed and showed effect of
antihypertensive, immunomodulatory activity, anti-cancer and calcium binding
ability. Fermented milk products are reported to contribute to human health
through several mechanisms LAB are in the first rank of listed organisms as
species used in probiotic preparation (Widyastuti, 2014).
Fermentation processes are believed to have been developed over the years
by women, in order to preserve food for times of scarcity, to impart desirable
flavour to foods, and to reduce toxicity. Fermentation has enabled our ancestors in
temperate and cooler regions to survive winter season and those in the tropics to
survive drought periods, by improving the shelf-life and safety of foods and
beverages. Fermented foods are treasured as major dietary constituents in
numerous developing countries because of their keeping quality under ambient
conditions - thereby contributing to food security - and because they add value,
enhance nutritional quality and digestibility, improve food safety, and are
traditionally acceptable and accessible (Marshall, 2011 : 2).
Anaerobic catabolic reactions are similar, although the electron acceptor
isn’t oxygen. The next example shows the fermentation of glucose to lactic acid.
C6H12O6 → 2 H3CCHOHCO2H + energy
In this case, one carbon (the methyl carbon of lactic acid) is reduced from the zero
oxidation state to –3 while another carbon (the carboxyl carbon of lactic acid)
gives up electrons and goes from an oxidation state of zero to +3. In this example,
the electron acceptor and electron donor are located on the same molecule, but the
principle remains the same: One component is oxidized and one is reduced at the
same time The general reaction accounts for the fact that in some systems,
something other than water supplies the reducing equivalents. For example,
bacteria living in deep-sea thermal vents can apparently use hydrogen sulfide
(H2S) as a source of reducing equivalents to synthesize glucose from carbon
dioxide dissolved in the seawater (Schmidt, 2000 : 48-49).
Adsorption of phage particles to bacterial cells is the initial step of phage
infection. While 75% adsorption occurred in 10 min, 96% adsorption occurred in
30 min. Adsorption is not only dependent on the presence of specific receptors on
the cell surfacebut can also depend on the presence of certain cations in the media.
Bacteriophages usually require higher concentrations of divalent cations, such as
calcium or magnesium, at some stage of their infection cycle than the
concentration required for the growth of host cellsreported that calcium ions were
required for the penetration of phage genomes into the host cells of Lactobacillus
casei (Z. Lu, 2003).
Soy sauce has been made for centuries by traditional methods, and
consumed as the source of protein and vitamins. Soy sauce contains essential
amino acids such as Valine, Tryptophan, Lysine, and Histidine and also contains
vitamins (especially vitamin B6) and antioxidants (isoflavones). Fermentation
increases protein content, eliminates trypsin inhibitors, and reduces the peptide
size in soybean meals. These effects of fermentation might make soy foods more
useful in human diets as a functional food and benefit livestock as a novel feed
ingredient. We recommend further studies relating to screening and selecting
bacteria strains as well as preservation condition (Minh, 2014).
In sourdoughs, flavour-active compounds are produced by LAB and yeasts
individually and through their interactions. Heterofermentative LAB mainly
produce ethyl acetate and certain alcohols and aldehydes, whereas
homofermentative LAB synthesise diacetyl and other carbonyls. In contrast, iso-
alcohols are products of yeast fermentation but may contribute little towards final
bread flavour. Interactions between microbial strains and ingredients,
fermentation temperature, pH and leavening time have all been evaluated. Further
research should be conducted on optimization of fermentation process in wheat
bread production by varying biotechnical parameters, levels of
emulsifiers/improvers including lactose, milk solids andoxidants. Effects of dried
mixed cultures of yeasts-LAB also need further attention (Rehmah, 2006).
The disaccharidases—maltase, sucrase-isomaltase (a bifunctional enzyme
catalyzing hydrolysis of sucrose and isomaltose), lactase, and trehalase—are
located on the brush border of the intestinal mucosal cells where the resultant
monosaccharides and others arising from the diet are absorbed. In most people,
apart from those of northern European genetic origin, lactase is gradually lost
through adolescence, leading to lactose intolerance. Lactose remains in the
intestinal lumen, where it is a substrate for bacterial fermentation to lactate,
resulting in discomfort and diarrhea (Murray, 2003 : 475).
The transition from a fossil fuel-based economy to a biofuel-based
economy depends on the improvement of biofuel yields via the successful
development of suitable microorgansims capable of efficiently fermenting a
variety of sugars while simultaneously displaying tolerance to high end product
concentrations. Cellulose represents an attractive feedstock for biofuels
production because of its abundance, cost and ability to be efficiently degraded by
cellulolytic bacteria. Metabolic engineering of bacterial and yeast species has
already demonstrated that the construction of novel strains using recombinant
DNA technologies can confer advantageous traits in regards to biofuels
production. Success, however, will be dependent upon design decisions based on
a detailed understanding of the extremely complex genetic, enzymatic,
and thermodynamic mechanisms that direct carbon flow. In combination
with other strategies including (meta)genomics, biodiversity studies and systems
biology, metabolic engineering is a promising approach to the improvement of
biofuel yields and the establishment of renewable, non-polluting energy
sources (Carere, 2008).
Flat bread is made mostly by traditional methods in Turkey, Middle East
and Northern African countries. Some types of the Turkish flat bread are; lavas,
yufka, pide, bazlama and gomme. Flat breads are made either leavened or
unleavened. They are also divided into two major groups: one (single) layered and
two (double) layered. Double layered flat breads are leavened with baker’s yeast
or sourdough. Single layered flat breads are made as both leavened and
unleavened (D. Gocman, 2009).
These results are consistent with the statements of Valerio et al. Saying
that raw materials used to produce bread represent a rich source of sporeforming
bacteria; therefore their microbiological quality should be monitored before use.
Other measures that can be taken in bread factories for preventing bread rope
spoilage refers to fast cooling of bread after baking, correct packaging of cooled
bread in clean packaging materials and keeping sanitary conditions along the
entire production process (Rumeus, 2013).
The very impressive scientific and technological developments that have
been made with lactic acid bacteria over the past number of years are likely to
Beuchat, relieve many of the bottlenecks encountered with their full and efficient
application in food fermen tations in the near future. The availability of in
formation regarding the genetic blueprint of these bacteria (the genome sequence
of a strain of Lactococcus has already been determined and those of several other
lactic acid bacteria will be available in the near future) will also provide
new applications that are likely to benefit both the producer and
consumer (Caplice, 1999).
D. APPARATUS AND CHEMICALS
1. Apparatus
a. Drop pipette 10 units
b. Drop plate 1 unit
c. Stopwatch 1 unit
d. Big test tube 12 units
e. Test tube rack 3 units
f. Beaker 100 ml 4 units
g. Funnel 1 unit
h. Stirrer 2 units
i. Spoon 2 units
j. Beaker 400 ml 1 unit
k. Beaker 1000 ml 1 unit
l. Bunzen burner 1 unit
m. Wood clamp 1 unit
n. Tripod and gauze 1 unit
o. Drop pippette 8 units
p. Measure glass 10 ml and 50 ml 1 unit
q. Small test tube 3 units
r. Spray bottle 1 unit
s. Rough and soft cloth 1 unit
2. Materials
a. Bread yeast (Saccharomyces cerevisiae)
b. Tape yeast
c. Tempe yeast (Rhizopusoligosporus)
d. Starch 1 % (C6H12O6)n
e. Glucose (C6H12O6)
f. Fructose 5% (C6H12O6)
g. Mannose 5% (C6H12O6)
h. Galactose 5% (C6H12O6)
i. Iod 0.1% (I-)
j. Benedict/fehling reagent (AgNO3)
k. Cotton
l. Aquadest (H2O)
m. Tissue
n. Label
E. WORK PROCEDURE
1. Starch hydrolysis
a. Suspension of bread, tape and tempe yeast were prepared by dissolved little
part of spatula into beaker glass and into each yeast were filled by 25 mL of
aquadest.
b. Drop plate was filled by 5 drops of starch solution then was labeled 1-10
c. Hole 1, 2 and 3 of drop plate were added by 5 drops of bread yeast suspension,
hole 4, 5, and 6 were added 5 drops of tape yeast suspension and hole 8, 9 an
10 were added 5 drops of tempe yeast suspension. Hole 1 was added 5 drops of
aquadest as a control.
d. After 5 minutes, hole 1, 4 and 7were added a drop of iod 0,1 % and the color
change was noted.
e. After 10 minutes, hole 2, 5 and 8were added a drop of iod 0,1 % and the color
change was noted.
f. After 15 minutes, hole 3, 6 and 9were added a drop of iod 0,1 % and the color
change was noted.
2. Alcohol Fermentation
a. 15 big test tube were prepared
b. 5 ml of starch 1% solution was added in tube 1,2, and 3 than cotton were used
for close the tubes.
c. Other tubes were filled glucose, fructose and galactose were done the same
treatment.
d. Tube react was closed by cotton
e. Test tubes were sterillizated at 1100C
f. 3 test tube that contain starch were cooled in temperature room, tube 1st was
added 1 ml of bread suspention, tube 2nd was added 1 ml of tape yeast and tube
3rd was added 1 ml of tempe yeast.
g. Other tubes were filled glucose, fructose and galactose were done the same
treatment.
h. All test tubes were incubated in temperature room, than all test tubes was
checked there were carbon dioxide after 24 hours.
i. All test tube was checked the alcohol with opened the closer of tubes than
smell from mouth of tube.
j. Tube 1, 2, and 3 were tested with benedict/fehling test.
k. Heat the react tube that filled of starch.
F. OBSERVATION RESULT
1. Hydrolisis of Starch Test

NO ACTIVITY RESULT

1 Some bread yeast (Brown) + 25 ml of Turbid solution

H2O
5 drops of amylum (Colorless) + 5 drops
of berad yeast suspense (white) Dark blue

a. Hole 1 (5 minutes)
Color less solution + 1 drop of iod 0.1%
(yellow)
Dark blue

b. Hole 2 (10 minutes)

Colorless solution + 1 drop of iod 0.1%
(yellow)
Dark blue

c. Hole 3 (15 minutes)

Colorless solution + 1 drop of iod 0.1%
(yellow)
2 Some tapai yeast ( white) + 25 ml of Turbid solution with
H2O precipitation
5 drops of amylum (colorless) + 5 drops
of tapai yeast suspense (colorless)
a. Hole (4) 5 minutes Dark blue

Colorless solution + 1 drop of iod 0.1%

(yellow)

Dark blue
b. Hole (5) 10 minutes
Colorless solution + 1 drop of iod 0.1%
(yellow)

Dark blue
c. Hole (6) 15 minutes
Colorless solution + 1 drop of iod 0.1%
(yellow)
3 Some tempe yeast ( white) + 25 ml of Turbid solution
H2O
5 drops of amylum (colorless) + 5 drops
of tapai yeast suspense (colorless) Dark blue

a. Hole (4) 5 minutes

Colorless solution + 1 drop of iod 0.1%
(yellow)
Dark blue

b. Hole (5) 10 minutes

Colorless solution + 1 drop of iod 0.1%
(yellow)
Dark blue

c. Hole (6) 15 minutes

Colorless solution + 1 drop of iod 0.1%
(yellow)
2. Alcohol Fermentation

NO ACTIVITY RESULT

1 -5ml of starch 1% was potted Colorless solution

into tube 1,2,3
Colorless solution
-5ml of glucose 1% was
potted into tube 4,5,6 Colorless solution

-5ml of fructose 1% was Colorless solution

potted into tube 7,8,9
Colorless solution
-5ml of sucrose 1% was
potted into tube 10,11,12

-5ml of galactose 1% was

potted into tube 13,14,15
2. All tubes was closed by Hot solution and colorless
cotton and sterillization in solution
autoclaft until 10 minutes
(T=1100C)
3. All tubes was refrigerent in Colorless solution
temperature room
4. a. In tube Ist (starch, glucosse, colorless solution
fructose,galactose) was
added 1 ml of bread yeast.
b. In tube 2nd (starch,
glucosse,
colorlesssolution
fructose,galactose) was
added 1 ml of tape yeast.
c. In tube 3rd (starch,
 glucosse, colorless solution
fructose,galactose) was
added 1 ml of tempe yeast.
5. After incubation in room
temperature until 24 hours
a. Starch
Turbid solution and alcohol
 Tube I (bread)
Turbid solution and alcohol
 Tube II (Tape)
Turbid solution and alcohol
 Tube III (Tempe)
b. Glucose
 Tube I (bread) Turbid solution and not alcohol

 Tube II (Tape) Turbid solution and not alcohol

 Tube III (Tempe) Turbid solution and not alcohol

c. Fructose
 Tube I (bread) Turbid solution and not alcohol

 Tube II (Tape) Turbid solution and not alcohol

 Tube III (Tempe) Turbid solution and not alcohol

d. Sucrose
 Tube I (bread) Turbid solution and not alcohol

 Tube II (Tape) Turbid solution and not alcohol

 Tube III (Tempe) Turbid solution and not alcohol

e. Galactose
 Tube I (bread) Turbid solution and not alcohol

 Tube II (Tape) Turbid solution and not alcohol

 Tube III (Tempe) Turbid solution and not alcohol

6. For tube that contain starch

test with Benedict test
 Tube I Dark blue

 Tube II Dark blue

Dark blue
  Tube III
7. For tube that contain starch
test with Tollens test
 Tube I Silver precipitation

 Tube II Silver precipitation

Silver precipitation
 Tube III

G. DISCUSSION
1. Tes Hidrolisis Pati
Pada percobaan ini amilum akan dihidrolisis menjadi glukosa dengan
melakukan penambahan mikroba yaitu ragi roti, ragi tape, dan ragi tempe.
Sebelumnya ketiga ragi tersebut dibuat suspensi dengan cara melarutkan dengan
aquadest. Dalam pengujian digunakan plat tetes sebagai medianya. Kemudian plat
tersebut diberi no 1 sampai 10. Semua lubang diisi dengan amilum. Pada lubang
plat tetes 1,2,3 ditambahkan dengan suspensi roti, lubang 5,6,7 ditambah dengan
suspensi tape dan lubang 9,10,11 ditambah dengan suspensi tempe. 10 menit
pertama ditambahkan dengan iod untuk plat 1,5,9, selanjutnya 10 menit kedua
ditambahkan iod untuk plat 2,6,10 dan 10 menit berikutnya ditambahkan iod
untuk plat 3,7,11. Adapun tujuan dari penambahan iod adalah untuk mengetahui
adanya pembentukan kompleks amilum yang ditandai dengan warna biru.
Dari hasil pengamatan diperoleh semua plat menunjukkan warna biru
keunguan saat penambahan, hanya saja warna tersebut tidak bertahan lama yakni
hilang setelah beberapa saat. Hasil yang diperoleh sudah sesuai dengan teori,
dimana semakin lama waktu yang digunakan maka amilum semakin banyak
terhidrolisis oleh ketiga ragi tersebut dan warna sebenarnya yang dihasilkan yaitu
warna ungu atau agak pudar.
Pada saat penambahan ragi roti pada plat 1,2,3 semua menghasilkan warna
putih keruh. Hal ini menandakan bahwa ragi roti dapat menghidrolisis amilum
menjadi monosakarida ataupun oligosakarida. Pada plat 5,6 menghasilkan warna
larutan bening setelah penambahan ragi tape, namun pada plat 7 berubah menjadi
orange. Hal ini membuktikan bahwa ragi tape tidak dapat menghidrolisis amilum.
Pada plat 9,10 dan 11 menghasilkan warna larutan yang tidak berwarna. Hal ini
menandakan bahwa ragi tempe dapat menghidrolisis amilum menjadi
amilopektin, menurut persamaan reaksi :
Ragi Roti

Amilum
Ragi tape

Amilum
Ragi tempe

Amilum
2. Fermentasi alkohol
Pada percobaan ini bertujuan untuk mempelajari kemampuan
memfermentasi amilum, glukosa, fruktosa dan sukrosa oleh beberapa jenis
inokulum murni ragi roti, ragi tape dan ragi tempe sehingga menghasilkan alkohol
dan membebaskan gas karbondioksida.
Menyiapkan 12 tabung reaksi, setiap 3 tabung masing-masing diisi dengan
pati, galaktosa, fruktosa, glukosa dan sukrosa, kemudian ditutup rapat dengan
menggunakan kapas lalu disterilisasi pada suhu 110oC. Tujuan dari ditutup dengan
kapas yaitu mencegah adanya O2 dalam tabung pada saat sterilisasi. Sedangkan
sterilisasi berfungsi untuk agar mikroorganisme yang terdapat dalam tabung dapat
mati sehingga tidak mengganggu proses fermentasi yang akan dilakukan oleh
ketiga jenis ragi tersebut. Adapun tujuan dari suhu 1100C untuk mematikan
mikroorganisme yang dapat mengganggu proses fermentasi.
 Setelah mencapai suhu 1100C, tabung didinginkan pada suhu kamar,
kemudian ketiga tabung reaksi yang berisi amilum masing-masing ditambahkan
dengan ragi roti, ragi tape dan ragi tempe. Melakukan perlakuan yang sama untuk
masing-masing tabung terhadap glukosa, fruktosa, dan sukrosa. Melakukan
inkubasi selama 24 jam terhadap masing-masing tabung karena proses dari
fermentasi dar ragi yang digunakan membutuhkan waktu yang cukup lama
sehingga dilakukan inkubasi. Adapun perlakuan didinginkan agar bakteri yang
disuntikkan ke dalam larutan tidak mati disebabkan suhu yang terlalu tinggi.
Berdasarkan hasil pengamatan diperoleh bahwa pada tabung yang berisi
amilum berbau alkohol dan terdapat gas CO2. Hal ini menandakan bahwa pada
amilum terjadi fermentasi alkohol. Hal ini tidak sesuai dengan teori bahwa ragi
roti (Saccharomyces cerevisae) tidak dapat mengfermentasi amilum menjadi
alkohol (Tim Dosen Biokimia, 2016: 1) dengan reaksi :

Ragi roti

Secara teori ragi tape dapat mengkonversi pati/amilum menjadi glukosa

dan gula-gula yang jauh lebih sederhana. Didalam kapang terdapat enzim amilotik
sehingga dapat memecah amilum menjadi gugus yang sederhana. Proses ini sering
disebut sakarifikasi. Lalu khamir akan merubah gula-gula sederhana tersebut
menjadi alkohol. Begitu pula pada ragi roti dan tempe yang dapat memecah pati
menjadi alkohol dan karbondioksida. Penyebab dari penyimpangan ini yaitu
adanya gangguan bakteri lain pada saat proses fermentasi berlangsung ataupun
disebabkan oleh rusaknya bahan akibat adanya kontaminasi dengan zat lain atau
udara luar.
Pada tabung berisi sukrosa hanya ragi tempe, ragi roti dan ragi tape tidak
menghasilkan bau dan tidak terdapat gas CO 2. Hal ini tidak sesuai dengan teori
yang menyatakan bahwa ragi roti (khamir) dapat memfermentasi glukosa menjadi
alkohol dan membebaskan gas CO2. Pada tabung yang berisi glukosa ragi tape,
ragi roti, dan ragi tempe tidak menghasilkan bau alkohol dan tidak terdapat gas
CO2. Hal ini tidak sesuai dengan teori dimana ragi roti (khamir) dapat
memfermentasi glukosa menjadi alkohol sambil membebaskan gas CO 2. Pada
tabung yang berisi fruktosa menghasilkan uji negatif dimana semua tabung yang
berisi ragi roti, ragi tape, dan ragi tempe tidak menghasilkan bau alkohol dan tidak
terdapat gas CO2.
Untuk tabung yang berisi amilum dibagi dua dan dilakukan uji benedict
serta uji tollens untuk pengujian gula-gula pereduksi pada amilum. Ketiga tabung
ditambahkan dengan larutan benedict menghasilkan warna biru kemudian
dipanaskan larutan tetap berwarna biru dan tidak terdapat endapan. Percobaan ini
tidak sesuai dengan teori dimana pada uji benedict diperoleh endapan merah bata.
Hal ini disebabkan larutan yang digunakan kurang baik akibat adanya kontaminasi
dengan zat lain atau udara luar. Begitupun ketiga Meurut persamaan reaksi :

Uji pereaksi fehling

Uji Pereaksi Tollens

H. CONCLUSSION AND SUGGESTION
1. Conclussion
From the experiment result can be concluded:
a. Bread yeast can hydrolyze glucose into alcohol while tape yeast and tempe
yeast can hydrolyze starch into glucose.
b. Bread yeast can ferment the glucose into alcohol and the CO2 of the host can
not ferment the starch into alcohol and CO2.
2. Suggestion
a. For assistent, I hope protec the apprentice when doing experiment.
b. For laboratorium, I hope can prepared test tube to fermentation experiment,
because very hard find out test tube.
c. For next apprentice to carefull when closed the test tube with cotton.

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