antioxidant activity of milk fermented with lactic acid bacteria from different fruits universiti sains

67 Pages·2015·2.22 MB·English

ANTIOXIDANT ACTIVITY OF MILK FERMENTED WITH
LACTIC ACID BACTERIA FROM DIFFERENT FRUITS

MARYAM A. S. ABUBAKR

UNIVERSITI SAINS ISLAM MALAYSIA
PERP/UR/1001

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
DECLARATION OF THESIS/ UNDERGRADUATE PROJECT PAPER AND COPYRIGHT
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

Author’s Full Name:
Maryam A. S. Abubakr

Student’s Number:
4090088

Title:
Antioxidant activity of milk fermented with lactic acid bacteria from different
fruits

Academic Session:
2012/2013

I hereby declare that the work in this thesis/ project paper is my own except for quotations and
summaries which have been duly acknowledged.

I acknowledged that Universiti Sains Islam Malaysia reserves the right as follows:

1. The thesis/ undergraduate project paper is the property of Universiti Sains Islam
Malaysia.

2. The library of Universiti Sains Islam Malaysia has the right to publish my thesis/
undergraduate project paper as online open access (fulltext) and make copies for the
purpose of research or teaching and learning only.

.......................................... ..........................................
(Student No: 4090088) (Signature of Supervisor)

(Pasport No: 996111 ) __________________________________

Date: Date:
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AUTHOR DECLARATION

هتاكربو الله ةوحرو نكيلع ملاسلا
نيحرلا نوحرلا الله نسب

I hereby declare that this thesis is my original work except for quotations and summaries
which have been dully acknowledged.

Date: JUNE 2013

Signature :

Name : Maryam A. S. Abubakr

Matric No. : 4090088

Address : 5-5A-18 JALAN JATI 1
DESA JATI BANDAR
BARU NILAI, 71800

NILAI, NEGERI
SEMBILAN.
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BIODATA OF AUTHOR

Maryam A. S. Abubakr (M4090088) was born on the 22th August 1977. She previously
was a student of Seventh of April University Libya and obtained Bachelor degree in
Botany 1998 and Master degree in Botany 2008 from Faculty of Sciences. She is at
present a PhD student of USIM in Science and Technology.
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APPROVAL

This thesis entitled “Antioxidant Activity of Milk Fermented with Lactic Acid
Bacteria from different fruits” submitted to the Faculty of Science and Technology,
USIM and was accepted as fulfillment of the requirements to the Doctor Philosophy of
Science and Technology.

ZAITON HASSAN, Ph.D,
Faculty of Science and Technology,
Universiti Sains Islam Malaysia

Date: JUNE 2013
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ACKNOWLEDGEMENTS

In the name of Allah, the most Gracious, the most Merciful.

Alhamdulillah, all praises to Allah SWT for giving me the strength to complete this study.

A special gratitude and sincere appreciation goes to Associate Prof. Dr. Zaiton Hassan,
my supervisor for this thesis for her guidance, generous advises, helpful comments,
patience and great concern from the beginning till the end of study, I consider myself very
lucky to be given this honor to work with her.

Not forgetting my love and thankfulness to parents, husband, sisters and brothers for their
understanding, strong supports and advices.

I also would like to thank my friends Mss Ghalia Eswai and Mss. Nagea Abdalsadiq for
their help and advises. My thanks of extend to Mr. Fredy Kesnawan Shah bin Abdul
Kahar and Mr. Mazlan for their outstanding help and support.

Thank you.
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ABSTRAK
Peptida bioaktif boleh dihasilkan daripada protein susu melalui fermentasi dengan
bakteria asid laktik (LAB). Dalam kajian ini LAB telah dipencikan daripada sampel
makanan yang berbeza. Antara 300 diasingkan LAB, 205 telah menunjukkan zon jernih
pada agar MRS-CaCO yang di ubahsuai, katalase negatif dan Gram positif, adalah dikira
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sebagai LAB dan digunakan untuk kajian ini. Dua puluh daripada 205 isolat yang
menujukkan aktiviti proteolitik pada agar susu skim menghasilkan peptida dalam
hidrolisat susu skim (SMH) dan menunjukkan aktiviti mengikat radikal bebas denagn
kadar dari 11 ke 50.8% (v/v) selepas 24 ke 72 jam fermentasi, masing masing,
sebagaimana yang ditentukan dengan kaedah 1,1-diphenyl-2-picrylhyrazyl (DPPH).
Aktivit pengkelat ferros (FCA) SMH menunjukkan nilai yang sama pada EDTA selepas
24 jam fermentasi tetapi menurun selepas 72 jam badi semua isolate LAB dengan nilai
antara 10.3 ke 97.6% (v/v) untuk 24 ke 72 jam masing-masing. Nilai IC DPPH SMH-
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Lactobacillus plantarum1 (SMH1) adalah lebih tinggi (2.92 mg/ml) daripada yang
Leuconostoc mesenteroides (SMH2) dan Lactobacillus plantarum ATCC8014 (SMH3)
8.81 dan 4.53 mg/ml, masing-masing. Nilai IC FCA SMH1 dan SMH2 adalah 0.46 dan
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0.69 mg/ml, masing-masing lebih besar daripada SMH3 (IC bernilai 0.74 mg/ml) tetapi
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lebih rendah daripada EDTA. Semua isolat menunjukkan kuasa penurun (RP) yang lemah
berbanding asid askorbik. SMH2 dan SM1 yang di-isolat daripada sumber bukan tenusu
mempunyai sifat probiotik dan antipengoksida yang boleh dimanafaatkan oleh pengguna.
Aktiviti proteolitik oleh SMH sangat dipengaruhi (p ≤ 0.05) oleh strain LAB yang
digunakan. SMH1 merencat pembiakan sel kanser lebih tinggi daripada SMH2 dan
SMH3 dengan nilai 20.63, 26.32 dan .63%, masing-masing pada kepekatan SMH 200
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µg/ml berbanding dengan sel kawalan yang tidak dirawat. Komposisi asid amino untuk
semua SMH adalah kaya dengan asid glutamik, prolin, lysin dan leusin dengan perbezaan
yang ketara (P ≤ 0.05). Walaubagaimanapun, jumlah asid amino hydrofobik pada SMH2
adalah lebih tinggi (13.98%) daripada SMH3 dan SMH1 (12.72% dan 8.23%), masing-
masing. Aromatik asid amino bersama asid amino hidrophobik menyumbang kepada
aktiviti antipengoksida. Ekstrak protein mentah SMH yang dihasilkan oleh tiga isolate
LAB telah di separa bersihkan daripada mendakan ammonium sulfat dan dialisis. Peptida
daripada bahagian yang paling aktif diambil daripada fasa songsang prestasi tinggi cecair
kromatografi (RP-HPLC) telah disusun berturutan menggunakan elektrospray pengionan
jisim spektometer (ESI-MS/MS). Aplikasi agen antikeperangan adalah kaedah yang
paling efektif untuk mengawal tindakbalas keperangan dalam buah –buahan dan sayuran.
Oleh itu kesan agen antikeperangan (asid askorbik, acid citrik, SMH1, SMH2 dan
SMH1+SMH2) keatas kepingan buah pear Cina dan kepingan ubi kentang telah dikaji.
Kajian meunjukkan kepingan pear Cina dan kentang yang dirawat dengan gabungan
SMH1+SMH2 merencat tindakbalas keperangan setanding dengan asid askorbik seperti
yang ditunjukkan oleh penurunan jumlah sebatian fenol. Oleh itu adalah boleh
dirumuskan bahawa isolate LAB tempatan mempuyani potensi digunakan untuk
menghasilkan peptida dalam SMH dan mempuyai aktiviti antioksida dan antikeperangan,
dwi fungsi bioaktif peptida yang akan berguna dalam formulasi makanan berfungsi.
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ABSTRACT
Bioactive peptides can be generated from milk protein by fermentation with lactic acid
bacteria (LAB). In this study LAB were isolated from different food samples. Among 300
LAB isolates, 205 isolates showed clear zone on modified MRS-CaCO agar, catalase
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negative and Gram positive, that were considered as LAB and used for this study. Twenty
out of 205 isolates that showed proteolytic activity on skim milk agar produced peptides
in skim milk hydrolysate (SMH) and show free radical scavenging activity ranging from
11 to 50.8% (v/v) after 24 to 72 h of fermentation, respectively as determined by 1,1-
diphenyl-2-picrylhydrazyl (DPPH) method. Ferrous chelating activity (FCA) of SMH
showed similar values for EDTA after 24 h fermentation but decreased after 72 h for all
LAB isolates with values between 10.3 to 97.6% (v/v) for 24 to 72 h, respectively. The
IC DPPH value of SMH-Lactobacillus plantarum1 (SMH1) was higher (2.92 mg/ml)
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than that of Leuconostoc mesenteroides (SMH2) and Lactobacillus plantarum
ATCC8014 (SMH3) 8.81 and 4.53 mg/ml, respectively. The IC FCA values of SMH1
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and SMH2 were 0.46 and 0.69 mg/ml, respectively greater than SMH3 (IC value 0.74
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mg/ml) but lower than EDTA. All isolates showed poor reducing power (RP) compared
to ascorbic acid. L. mesenteroides and L. plantarum1 isolated from non-dairy sources
have probiotic and antioxidative properties which could benefit consumers. The
proteolytic activity of SMH was significant affected (p ≤ 0.05) by the strains of LAB
used. The SMH1 was higher inhibit the cell proliferation of cancer cells than SMH2 and
SMH3 with values 20.63, 26.32 and 32.63%, respectively at concentration of SMH 200
µg/ml compared with the untreated control cells. The amino acid compositions for all
SMH were significantly (P ≤ 0.05) rich in glutamic acid, proline, lysine and leucine.
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However, the total hydrophobic amino acids content in SMH2 was higher (13.98%) than
SMH1 and SMH3 (12.72%, and 8.23%), respectively. Aromatic amino acids along with
hydrophobic amino acids contribute to antioxidant activity. The crude protein extracts of
SMH generated by three LABs isolates were partially purified by ammonium sulfate
precipitation and dialysis. The peptides from most active fractions collected by reverse
phase high-performance liquid chromatography (RP-HPLC) were sequenced by
electrospray ionization mass spectrometry (ESI-MS/MS). The application of
antibrowning agents is one of the most effective methods for controlling the browning
reaction in fruits and vegetables. Therefore, the effect of antibrowning agents (Ascorbic
acid, Citric acid, SMH1, SMH2 and SMH1+SMH2) on Chinese pear and potato slices
was investigated. The results indicate that the slices of Chinese pear and potato treated
with combining SMH1+SMH2 inhibited browning reaction comparable to ascorbic acid
as shown by reduction of total phenol content. Therefore, It can be concluded that this
local LAB isolates have the potential to be used to generate peptides in SMH and have the
antioxidant and antibrowning activities, a dual function of bioactive peptides that would
be useful in the formulation of functional foods.