Fermentation

APPROVAL SHEET

Complete report of Biochemical Experiment with title “Fermentation” which has made by   :

            Name               : Fahruddin Ilham Nur

            Reg. number    : 091304182

            Class / Group  : ICP  / II (Second)

            Have been checked and correctedby assistant and assistant coordinator and it was acceptable.

                                                                             Makassar, on October            2011

 Known by,                                                                                  Assistant

Lecturer of Responsibility

Rina Arizanti S.Si                                                     Resi Agestia Waji S.Si, M.Si

A.      Title of Experiment

          FERMENTASI

B.  Purpose of experiment

To learning fermentation ability of starch, glucose, fructose, mannose, galactose, by some inoculume bread yeast, tape, yeast and soy yeast (Rhizopus oligosporus)

C.  Preview Literatures

By tradition, lactic acid bacteria (LAB) are the most commonly used microorganisms

for preservation of foods. Their importance is associated mainly with their safe

metabolic activity while growing in foods utilising available sugar for the production

of organic acids and other metabolites. Their common occurrence in foods and feeds

coupled with their long-lived use contributes to their natural acceptance as GRAS

(Generally Recognised As Safe) for human consumption (Aguirre & Collins, 1993).

However, there are many kinds of fermented foods in which the dominating

processes and end products are contributed by a mixture of endogenous enzymes and

other microorganisms like yeast and mould. Very often, a mixed culture originating

from the native microflora of the raw materials is in action in most of the food

fermentation processes. However, in an industrial scale a particular defined starter

culture, which has been developed under controlled conditions, is of first preference

so that the qualities of the finished product could be consistently maintained day

after day. Moreover, modern methods of gene-technology makes it possible for the

microbiologists to design and develop starter cultures with specific qualities (Brandy,2009 ).

                By tradition, lactic acid bacteria (LAB) are the most commonly used microorganisms

for preservation of foods. Their importance is associated mainly with their safe

metabolic activity while growing in foods utilising available sugar for the production

of organic acids and other metabolites. Their common occurrence in foods and feeds

coupled with their long-lived use contributes to their natural acceptance as GRAS

(Generally Recognised As Safe) for human consumption (Aguirre & Collins, 1993).

However, there are many kinds of fermented foods in which the dominating

processes and end products are contributed by a mixture of endogenous enzymes and

other microorganisms like yeast and mould. Very often, a mixed culture originating

from the native microflora of the raw materials is in action in most of the food

fermentation processes. However, in an industrial scale a particular defined starter

culture, which has been developed under controlled conditions, is of first preference

so that the qualities of the finished product could be consistently maintained day

after day. Moreover, modern methods of gene-technology makes it possible for the

microbiologists to design and develop starter cultures with specific qualities

 (Yean, 2011).

                Metabolism refers to the biochemical assimilation (in anabolic pathways) and dissimilation (in catabolic pathways) of nutrients by a cell. Like in other organisms, in yeast these processes are mediated by enzymic reactions, and regulation of the underlying pathways have been studied in great detail in yeast. Anabolic pathways include reductive processes leading to the production of new cellular material, while catabolic pathways are oxidative processes which remove electrons from substrates

or intermediates that are used to generate energy. Preferably, these processes use NADP or NAD, respectively, as co-factors. Although all yeasts are microorganisms that derive their chemical energy, in the from of ATP, from the breakdown of organic compounds, there is metabolic diversity in how these organisms generate and consume energy from these substrates. Knowledge of the underlying regulatory mechanisms is not only valuable in the understanding of general principles of regulation but also of great importance in biotechnology, if new metabolic capabilities of particular yeasts have to be exploited  (Sabirin, 1994).

                In this section of the course we will cover the primary fermentation, the conversion of sugar to ethanol, which is the foundation of the transformation of grapes into wine. The first lecture will cover the basic biology of the yeast Saccharomyces. Subsequent lectures will cover all aspects of fermentation management, and the problems that can arise. Principle among these problems is off-character production and slow or incomplete fermentations. The alcoholic fermentation is conducted by yeast of the genus Saccharomyces. The two common species involved are S. cerevisiae and S. bayanus. These two species are closely related, and the subject of a continuing debate among taxonomists as to whether they constitute separate species or races of the same species. Saccharomyces converts the glucose, fructose and sucrose found in grape must and juice into ethanol via the process of fermentation. In fermentation, an organic

compound, in this case acetaldehyde, serves as terminal electron acceptor. This leads

to the production of ethanol (Lecturer, 2011).

Generally, a significant increase in the soluble fraction of a food is observed during fermentation. The quantity as well as quality of the food proteins as expressed by

biological value, and often the content of watersoluble vitamins is generally increased, while the antinutritional factors show a decline during fermentation (Paredes-López & Harry, 1988). Fermentation results in a lower proportion of dry matter in the food and the concentrations of vitamins, minerals and protein appear to increase when measured on a dry weight basis (Adams, 1990). Single as well as mixed culture fermentation of pearl millet flour with yeast and lactobacilli significantly increased the total amount of soluble sugars, reducing and non-reducing sugar content, with a simultaneous decrease in its starch content (Khetarpaul & Chauhan, 1990). Combination of cooking and fermentation improved the nutrient quality of all tested sorghum seeds and reduced the content of antinutritional factors to a safe level in comparison with other methods of processing (Obizoba & Atii, 1991). Mixed culture fermentation of pearl millet flour with Saccharomyces diastaticus, Saccharomyces cerevisiae, Lactobacillus brevis and Lactobacillus fermentum was found to improve its biological utilisation in rats (Winarno,2004)

D.  Tools and Reagent

a.       Tools:

1.     est tube 1 unit

2.      pipette

3.      test tube 12 units

4.      spatula 1 unit

5.      test tube rack  2 unit

6.      stirring bar 1 unit

7.      beaker glass 50 ml 3 units

8.      sprayer bottle

9.      Beaker glass 1000 ml 1 unit

10.  Stopwatch

11.  azbestoz gavze, three pot 1 unit

12.  volumetric glass 10 ml and 50 ml 2 unit

b.      materials:

1.      bread, tape, soy yeast powder (Saccharomycess), (Rhizopus)

2.      cotton

3.      iod solution 0,1 % (Kl)

4.     starch 1% (C12H24O13)

5.      fructose 5%

6.      Glucose

7.      Galactose 5%

8.      Sucrose

9.      bennedict reagent

10.  Aquadest

11.  fehling reagen

12.  tollens reagent

13.  tissue

14. Aluminium Voil

15. matches

E.   Work Procedures

a.    hydrolizis starch test

1.      made suspension of bread yeast, tape yeast and tempe yeast by dilute powder (starch) into 25 ml water.

2.      prepared drop plate and labelled in each tubes of plate 1-10

3.      filled each tubes by 1 ml of starch

4.      holes 1 filled by aquadest 1 ml and iod 1%

5.      hole 2,3,4 added 1 ml bread yeast

6. hole 5,6,7 added 1 ml tape yeast

7. hole 8,9,10 added 1 ml tempe yeast

8. after 5 minutes plate 2,5,8 added iod 1 % 1 drop

9. after 10 minutes plate 3,6,9 addedio 1% 1 drop

10. after 15 minutes plate 4,7,10 added iod 1 % 1 drop

11. observed the color formed.

b.    alcohol fermentation

1.      Meprepared 15 test tube

2.      filled 3 test tube by starch solution 5 ml and done some treatment for fructose, glucose, galactose, and sucrose.

3.     covered by cotton

4.      sterillized each solution in all tubes in autoclave in 110⁰ during 10 minutes.

5.      took out tubes and cold in room temperature.

6.     added 1 ml bread yeast in one of tube filled bread yeast, glucose, fructose, sucrose and galactose.

7.      done some treatments for tape and tempe yeast, in each of samples.

8.      cold all tubes in room temperature24 hours.

9. checked presence CO2 by shake tubes after kept 24 hours and also smell the alcohol.

10. tested bennedict and tollens for tubes filled starch.

F.   Result

1.      starch hydrolized test

a.- 1/10 spoon Saccharomycess cerevisiae + 25 ml H₂O            suspention (turbid).

     -1/10 spoon Rhyzopus oligosporus + 25 ml H₂O                suspention (turbid).

     - 1/10 spoon tape yeast + 25 ml H₂O                suspention (turbid).

b. -  2,3,4 plat (1 ml starch)+ 1 ml Rhyzopus suspention                 transparant

        solution and turbid.

     - 5,6,7 plat (1 ml starch)+ 1 ml Saccharomycess suspention                 transparant

        solution and turbid.

     - 8,9,10 plat (1 ml starch)+ 1 ml tape yeast suspention                 transparant

        solution and turbid.

c. -after 5 minutes

     2,5,8 plat (Transpatant O ) + 1 drops iod 1 % blue

                -plat 2 = young purple

                -plat 5 = yaoung purple

                -plat 8 = young purple

   -after 10 minutes

    3,6,9 plat (Transpatant O ) + 1 drops iod 1 % blue

                -plat 3 = rather purple

                -plat 6 = rather purple

                -plat 9 = rather purple

   -after 15 minutes

    4,7,10 plat (Transpatant O ) + 1 drops iod 1 % blue

                -plat 4  = old purple

                -plat 7  = yold purple

                -plat 10 = old purple

2.      Alcohol Fermentation

a.    5 ml amylum solution 1 % Sterillised + 1 ml (tube 1,2,3). Saccharomycess cerevisie

      (tube 1)+ 1 ml Rhyzopus oligosporus (tube 3)+ 1 ml tape yeast (tube 2).

b.    Glucose Sterillised + 1 ml (tube 1,2,3). Saccharomycess cerevisie (tube 1)+ 1 ml

       Rhyzopus oligosporus (tube 3)+ 1 ml tape yeast (tube 2).

c.    Sucrose Sterillised + 1 ml (tube 1,2,3). Saccharomycess cerevisie (tube 1)+ 1 ml

      Rhyzopus oligosporus (tube 3)+ 1 ml tape yeast (tube 2).

d.    Fructose Sterillised + 1 ml (tube 1,2,3). Saccharomycess cerevisie (tube 1)+ 1 ml

      Rhyzopus oligosporus (tube 3)+ 1 ml tape yeast (tube 2).

e. Galactose Sterillised + 1 ml (tube 1,2,3). Saccharomycess cerevisie (tube 1)+ 1 ml

      Rhyzopus oligosporus (tube 3)+ 1 ml tape yeast (tube 2).

After incubation in Autocave

Samples	Alcohol	Bubbles
Tube 1 : Glucose + Saccharomycess cerevisie Tube 2 : Glucose + tape yeast Tube 3 : Glucose + tempe yeast	Ö Ö Ö	- - -
Tube 1 : sucrose + Saccharomycess cerevisie Tube 2 : sucrose + tape yeast Tube 3 : Sucrose + tempe yeast	Ö - Ö	Ö - -
Tube 1 : fructose + Saccharomycess cerevisie Tube 2 : fructose + tape yeast Tube 3 : fructose + tempe yeast	Ö - -	- - -
Tube 1 : Galactose + Saccharomycess cerevisie Tube 2 : Galactose + tape yeast Tube 3 : Galactose + tempe yeast	- - -	- - -

Samples	Alcohol Smell	Bubble
Tube 1 = Amylum + Saccharomycess Tube 2 = Amylum + tape yeast Tube 3 = Amylum + Rhyzopus	Ö Ö Ö	- - -

Samples	Tollens	Fehling	Bennedict
Amylum 1 Amylum 2 Amylum 3	- - Silver mirror	- - -	Blue Blue Red bricked

G.  Discussion

Percobaan ini dilakukan untuk mengetahui kemampuan hidrolisis pati dalam menfermentasi glukosa, fruktosa, galaktosa , sukrosa, dan amilum oleh beberapa jenis inokulum dari ragi, tempe, tape dan roti. Dalam 1 gram ragi padat terdapat kurang lebih 10 milyar sel hidup berbentuk bulat dilindungi oleh dinding membrane yang berpori. Ragi tape berwujud padat bulat pipih, ragi roti berbentuk butiran, sedangkan ragi tape berbentuk bubuk. Ragi roti dan ragi tape mengandung mikroorganisme yang sama yaitu Saccharomycess cerevisiae. Fungsi dari Saccharomycess cerevisiae yaitu mengubah glukosa menjadi alkohol dan CO2. Cepat berkembang biak dan tahan terhadap kadar alkohol yang tinggi. Sedangkan ragi tempe (Rhyzopus) dapat menfermentasi substrat dengan menghasilkan enzim terlebih dahulu.

                Pada tes hidrolisis pati, suspensi ragi ditambahkan pada tiap sampel dan di tambahkan iod 1% yang berfungsi untuk mengetahui adanya pembentukan senyawa kompleks amilum yang ditandai dengan warna ungu. Pembentukan kompleks (senyawa amilosa) pada waktu lebih lama (15 menit) menghasilkan warna ungu lebih pekat, hal ini desebabkan semakin lama waktu proses hidrolisis semakin maksimal karena sel-sel yang terdapat pada ragi akan rusak dan membrane luar sel tidak dapat menahan cairan (iod) keluar masuk sel. Ini menyebabkan warna biru dari iod masuk kedalam sel lebih banyak pada waktu lebih lama dibanding waktu 5 dan 10 menit. Sehingga warna yang ditunjukan lebih pekat. Sedangkan pada waktu 5 menit dan 10 menit, warna larutan tidak terlalu pekak, disebabkan sel-sel ragi tidak rusak seluruhnya, sehingga mampu menahan maksudnya iod pada kulit membrane. Perubahan warna dari amilum setelah ditambahkan ragi, dan iod akan menghasilkan rentan waktu yang berbeda. Semakin cepat hidrolisis maka semakin cepat perubahan warna. Penambahan ragi tape dan tempe tergolong cepat menghasilkan larutan biru pada amilum. Dari semua jenis ragi yang digunakan, menghasilkan uji positif terhadap kemampuan masing-masing ragi menhidrolisis amilum yang menghasilkan perubahan warna pada selang waktu yang berbeda.

                Pada fermentasi alkohol, semua sampel karbohidrat yang ada dalam tabung reaksi ditutup dengan kapas agar udara atau oksigen tidak masuk dalam tabung. Hal ini dilakukan karena dalam proses fementasi akan lebih baik jika tidak melibatkan oksigen ataub proses fermentasi tidak memerlukan oksigen. Selain itu, ragi juga dapat rusak jika dibiarkan terkontaminasi dengan udara luar akibat dari reaksi oksidasi terhadap udara luar.

                Selanjutnya semua sampel dimasukkan kedalam autoklaf, untuk disterilkan pada 110⁰c. Hal ini dilakukan karena pada suhu ini semua zat-zat pengganggu dapat dihilangkan. Setelah itu semua tabung didinginkan pada suhu kamar, karena pada suhu ini kerja enzim dapat optimaluntuk terjadinya fermentasi. Enzim disini berupa mikroorganisme yang terdapat dalam ragi, sehingga ragi tersebut ditambahkan pada suhu kamar. Setelah penambahan ragi, semua tabung ditutup kembali dengan kapas dan diinkubasi pada suhu kamar yang merupakan suhu optimal dari kerja enzim ini.

                Setelah masa inkubasi 24 jam, semua sampel pada tabung reaksi diamati adanya gas CO2 dengan cara menggoyang tabung reaksi, jika terdapat gelembung gas berarti positif adanya gas CO2 alam sampel. Kemudian mengamati juga adanya alkohol dalam sampel, dengan cara mencium mulut tabung, jika terdapat bau dari alkohol berarti sampel positif mengandung alkohol. Reaksi yang terjadi :

                Dari hasil percobaan, dari semua sampel hanya tabung sukrosa dengan penambahan ragi roti (Saccharomycess) yang menghasilkan gelembung (CO2) , selebihnya tidak menhasilkan. Hal ini disebabkan kesalahan praktikum yang tidak menggoyang-goyang tabung terlebih dahulu, sehingga gas tidak terbentuk. Sedangkan yang menunjukkan uji positif adanya alkohol yaitu pada sampel glukosa, sukrosa tabung 1, frukstosa tabung 1, dan amilum. Bau alkohol paling menyengat dimiliki oleh amilum. Hal ini disebabkan amilum tidak memerlukan waktu lama dalam menfermentasi. Saat diuji dengan reagent tollen, pati menunjukkan uji positif dengan membentuk cincin perak dan endapan merah saat penambahan bennedict. Hal ini menunjukkan bahwa pati mengandung gula pereduksi.

H. Conclussion and suggestion

Conclussion

1.       Starch hydrolysis indicated by color change become purple.

2.       Suitable yeast for hydrolysis is tape yeast and tempe yeast, due to needed shortest time to hydrolized.

3.       Yeast of bread, tape and tempe have ability to hydrolize sample to produce CO2 gas and alcohol.

Suggestion

For alcohol fermentation, of starch, better if incubation time is more than 24 hours in otder fermentation can running well to producing alcohol.

Bibliography

Brandy, 2009. Yeast. Htt://id.wikipedia@org/yeast.food category. Accessed @25

               December 2011.

Yean, 2011. Fermentation. http://www.id.biokimia/fermentation. accessed

              @ December  25^th 2011.

Sabirin. 1994. Organic Chemistry. Yogyakarta: UGM.

Lecturer. 2011. Penuntun Praktikum Biokimia. Makassar: FMIPA UNM.

Winarno. 2004. Food Chemistry and Nutrients. Jakarta : UI Press.

Translation of Discussion

The experiment was conducted to determine the ability of hydrolysis of starch in menfermentasi glucose, fructose, galactose, sucrose, and starch by several types of inoculum of yeast, tempeh, tape and bread. In 1 gram of solid yeast there are approximately 10 billion live cells rounded walls covered by a porous membrane. Yeast tape flat round are solid, granular baker's yeast, while the tape-shaped yeast powder. Yeast breads and yeast contain microorganisms of the same tape that Saccharomycess cerevisiae. The function of Saccharomycess cerevisiae that convert glucose into alcohol and CO2. Rapidly proliferating and resistant to high levels of alcohol. While yeast tempeh (Rhyzopus) can menfermentasi substrate with the enzyme first.
In the starch hydrolysis test, the yeast suspension was added to each sample and add 1% iodine that serves to detect the formation of complex compounds starch marked with purple. Complex formation (compound amylose) at longer time (15 min) resulted in more intense purple color, this is largely attributable the longer the maximum hydrolysis process because the cells contained in the yeast will be damaged and the outside of the cell membrane can not withstand fluid (IoD ) and out of the cell. This causes the blue color of iodine into the cells much more on a longer time than at 5 and 10 minutes. So the more intense the color indicated. While at the time of 5 minutes and 10 minutes, the solution color is not too deaf, because yeast cells are not damaged completely, so as to hold the point of iodine in the skin membrane. Change the color of the starch after adding yeast, and iodine will produce different vulnerable time. The faster the faster hydrolysis discoloration. The addition of yeast tape and tempeh quite quickly produce a blue solution on the starch. Of all the types of yeast used, resulting in a positive test for the ability of each yeast menhidrolisis starch resulting color change at different time intervals.
In alcoholic fermentation, all samples available carbohydrate in a test tube covered with cotton to allow air or oxygen into the tube. This is done because the process would be better fementasi not involve oxygen ataub fermentation does not require oxygen. In addition, the yeast can also be damaged if allowed to become contaminated with outside air due to oxidation of the outside air.
Furthermore, all samples included in the autoclave, sterilized at 1100c. This is done because at this temperature all intruders substances can be eliminated. After that all the tubes cooled to room temperature, because at this temperature the enzyme can optimaluntuk the fermentation. Enzyme here in the form of microorganisms contained in the yeast, so the yeast is added at room temperature. After the addition of yeast, all the tubes closed with cotton and incubated at room temperature which is the optimal temperature of these enzymes work.
After an incubation period of 24 hours, all samples in the test tubes have been observed CO2 by shaking the test tube, if there is a significant positive for the presence of gas bubbles of natural CO2 gas samples. Then observe also the presence of alcohol in the sample, by kissing the mouth tube, if there is a smell of alcohol means that the sample tested positive for alcohol. Reaction occurs:




From the experimental results, of all sample tubes only sucrose by the addition of yeast bread (Saccharomycess) which produces bubbles (CO2), the rest did not bring out. This is due to lab error is not rocking the first tube, so the gas is not formed. While indicating that test positive for the presence of alcohol in a sample of glucose, sucrose tube 1, frukstosa tube 1, and starch. The most stinging odor of alcohol held by starch. This is due to starch does not need much time in menfermentasi. When tested with a reagent tollen, starches showed a positive test by forming a ring of silver and red precipitate when added Bennedict. This suggests that the starch containing reducing sugars