Indonesian fermented

legumes: Modification of
phenolic compounds

D r. S i t i A i s ya h , M . S i .
Study Program of Chemistry
Universitas Pendidikan Indonesia
Overview
• General View of Food Fermentation:
• Indonesian Fermented Food
• Indonesian Legumes-based Fermented Food
• Tempe
• Oncom
• Kecap
• Tauco
• Fermented legumes and Modification of Phenolic Compounds
• Legumes Elicitation: Induction of Phytoalexins
General view:
Food Fermentation
Food Fermentation: Definition & Purpose
• One of the oldest and economical methods of producing and preserving food
• Fermented foods produced by people using their native knowledge from
locally available plant or animal sources either naturally or by adding starter
cultures containing functional microorganisms that modify the substrates
biochemically and organoleptically into edible products that are culturally and
socially acceptable to the consumers

Encyclopedia of Food Microbiology (Second Edition), 2014

Food Fermentation: Purpose
• Purpose:
• preserve food
• improve sensory quality (texture, flavor, aroma, color, elimination beany
flavor)
• enhance nutritive value (functional foods)

• Fermented food used as

• a main course
• a flavoring agent
• a soup base
• a food colorant
Food Fermentation: Process
• Traditional (small scale) vs modern (large scale) fermentation

• Submerged (involving water) vs solid state fermentation (free of water)

• Substrate:
• Plant: legumes, seeds, nuts, cereals, fruits, herbs, vegetables, tubers
• Animals: meat, milk, egg, fish
Xxx
Food Fermentation: Process
• Starter Cultures:
• Yeast: Saccharomyces cerevisiae, Candida stellata
• Fungi: Rhizopus sp., Neurospora sitophila, Aspergillus sp.
• Bacteria: (induce lactic acid bacteria, LAB) Lactobacilli, Pediococci,
Bacillus sp. Pseudomonas sp, Leuconostoc mesenteroides
• By using indigenous fermenter or by adding starter

Xxx
Indonesian Fermented Food
 Mandai

Tempoyak

Sayur asin
Acar

Picts sources: koki.id, Kompasiana, Pinterest

 Fruit- and Vegetable-based Fermented
Food
Regions of
Nature and consumption in
Foods Substrates uses Microorganisms Indonesia
Acar Vegetables Condiment Lactobacillus plantarum All regions

Sayur asin Vegetables Condiment Lactobacillus plantarum, L. confusus, West Java

L. curvatus, Leuconostoc mesenteroides,
Pediococcus pentosaceus
Tempoyak Flesh of Condiment Enterococcus gallinarum, Ent. faecalis, Sumatra
durian (Durio Oenococcus sp., Leuconostoc sp.,
zibethinus) Lactococcus sp., Pediococcus acidilactici,
Lactobacillus sp.
Mandai Inner part of Condiment Pediococcus pentosaceus, Lactobacillus Kalimantan
cempedak or plantarum, L. pentosus
jackfruit

Surono, I. S., 2016

 Gatot Growol

Tape singkong

Picts sources: Kulinerasik, Suarakonsumen, Pinterest

 Tuber-based Fermented Food
Regions of
Nature and consumption in
Foods Substrates uses Microorganisms Indonesia
Tape Cassava Snack Rhizopus oryzae, Muxor rouxii, West Java, Central
Aspergillus oryzae, Saccharomyces Java
cerevisiae, Endomycopsis burtonii,
Hansenula anomala,
Pediococcus pentosaceus.
Lactobacillus plantarum and
L. fermentum

Growol Cassava Snack Coryneform, Streptococcus, Bacillus, Yogyakarta

Actinobacteria, Lactobacillus, and yeast

Gatot Cassava Staple food Pediococcus pentosaceus, Yogyakarta

Saccharomyces sp. Lactobacillus
plantarum
Surono, I. S., 2016
 Brem Madiun

Brem Bali

Tape ketan

Picts sources: Net

 Cereal-based Fermented Food
Regions of
Nature and consumption in
Foods Substrates uses Microorganisms Indonesia
Tape Ketan Glutinous Snack Rhizopus oryzae, Muxor rouxii, West Java, Central
Hitam rice Aspergillus oryzae, Saccharomyces Java
cerevisiae, Endomycopsis burtonii,
Hansenula anomala,
Pediococcus pentosaceus.
Lactobacillus plantarum and
L. Fermentum

Brem Glutinous Snack, Rhizopus oryzae, Muxor rouxii, Central Java, Bali
rice beverages Aspergillus oryzae, Saccharomyces
cerevisiae, Acetobacter aceti

Surono, I. S., 2016

Dadih
Urutan

Picts sources: Travelingyuk, Oemahdekmong

 Meat- and Milk-based Fermented Food
Regions of
Nature and consumption in
Foods Substrates uses Microorganisms Indonesia
Dadih Buffalo milk Beverage Lactococcus lactis subsp. lactis, West Sumatra
Lactobacillus brevis, L. plantarum,
L. casei, L. paracasei, and Leu.
mesenteroides

Urutan Meat, pork Side dish L. plantarum, L. farciminis, obligate Bali

heterofermentative lactobacilli,
L. fermentum, L. hilgardii
P. acidilactici, P. pentosaceus

Surono, I. S., 2016

 Telur asin

Terasi

Peda

Picts sources: Lazada, Detik, Steemit

 Fish- and Egg-based Fermented Food
Regions of
Nature and consumption in
Foods Substrates uses Microorganisms Indonesia
Peda Fish Side dish Lactobacillus plantarum, L. curvatus, L. Java
murinus and Strepcocus thermophilus

Terasi Fish and Condiment Bacillus sp. and Sumatra, Java

shrimp Pseudomonas sp.

Telur asin Duck egg Side dish L. plantarum, L. casei subsp. All region
rhamnosus, Enterococcus gallinarum,
Pediococcus acidilactici

Surono, I. S., 2016

Legumes-based Fermented
Food
Legumes
• The most exploited plant families worldwide by human
• Domesticated for food and non-food purposes
• The third most species-rich among flowering plants, comprising approximately
19,325 species and 727 genera
• Three subfamilies: Caesalpinioideae, Mimosoideae and Papilionoidea, further
divided into 36 tribes
• The Papilionoideae is the largest subfamily with 28 tribes, 478 genera and
13,800 species, including economically important commodity legumes
 Edible legume
• Nutritive compounds: protein, dietary
fibre, complex carbohydrates, resistant
starch, vitamins and minerals
• Alkaloids, saponins, and phenolic
compounds (decrease the
bioavailability of some nutrients)

Pro-nutrients with positive health

effects contributing to the prevention
of diseases
 FAO categories Main use Production Species belonging to the FAO categories
categories (x103 tonnes) Scientific names Common names
Alfalfa N2 fixation n.aa Medicago sativa Alfalfa
Bambara beans Dry-grain 244 Vigna subterranea Bambara groundnut

Legumes Beans Dry-grain 23139

Phaseolus aconitifolius

Phaseolus acutifolius
Moth bean

Tepary bean

Phaseolus aureus Mungo bean

Phaseolus calcaratus Rice bean
Production of legume Phaseolus coccineus Scarlet runner bean
commodities in 2013. Phaseolus lunatus Lima bean
Phaseolus vulgaris Kidney bean
Source: Food and Agriculture Vigna angularis Adzuki bean
Vigna mungo Black gram
Organization of the United Vigna radiata Mung bean
Broad beans Dry-grain 3398 Vicia faba var. equina Horse bean
Nations (FAO) database Vicia faba var. major Broad bean
(http://faostat3.fao.org). Vicia faba var. minor Field bean

a n.a.: Data were not available. Chick peas Dry-grain 13102 Cicer arietinum Chick pea
Clover N2 fixation n.aa Trifolium spp. Clover
Cow peas Dry-grain 5718 Vigna unguiculata Cowpea
Groundnut Oil bearing 45225 Arachis hypogaea Groundnut
Lentils Dry-grain 4952 Lens esculenta Lentil
Lupins Dry-grain 786 Lupinus spp. Lupin
Peas Green-vegetable 10980

Pisum arvense Field pea

Pisum sativum Garden pea
Pigeon peas Dry-grain 4742 Cajanus cajan Pigeon pea
Pulses Dry-grain 5212
Canavalia spp. Jack bean
Cyamopsis Guar bean
tetragonoloba
Pachyrrhizus erosus Yam bean
Stizolobium spp. Velvet bean
Lablab spp. Lablab
Soybeans Oil bearing 276406 Glycine max Soybeans
Vetches Dry-grain 735 Vicia sativa Spring vetch
 Kecap

Tauco
Tempe

Oncom
Picts sources: Wikipedia, Net, Shoppe, Greenara
 Legume-based Fermented Food
Regions of
Nature and consumption in
Foods Substrates uses Microorganisms Indonesia
Tempe Soybeans Side dish Rhizopus oligosporus All regions

Oncom Soybean, Side dish Neurospora sitophila, R. West Java

peanut oligosporus

Kecap Soybean Condiment Aspergillus oryzae, A. sojae, R. oryzae, All regions

R. oligosporus

Tauco Soybean Condiment R. oligosporus, R. oryzae, A. oryzae, West Java

Lactobacillus delbrueckii, Hansenula
sp.

Surono, I. S., 2016

 • Washing & soaking soybeans
How to make tempe(h)? 1

• Precook by boiling, dehulling the

2 beans

• Soaking 12h soybean

3 (fermentation by LAB, pH 5)

• Boiling, washing, draining &

4 cooling the soybean

• Inoculating soybean
5 (fermentation by R. oligosporus)

• Pressing the fermented soybean

6
(Video from Trans7Official_YouTube)
• Incubating soybean 1-2 d, room
7 T
 How to make oncom? • Washed & boiled peanuts (dreg)
1 • Steamed tofu & casava dreg

• Mixing the ingredient

2

• Inoculating by Neurospora
sitophila/R. oligosporus
3

• Pressing the mixture

5

(Video from Trans7Official_YouTube)

• Incubating the mixture
6
 • Washing & Soaking soybeans
Tauco 1

• Boiling, dehulling, & washing

2 soybean

• Sun drying the soybean (half

3 dried)

• Natural fermentation 3d in
4 humid room

• Sun drying the fermented

5 soybean

• Soaking the fermented

6 soybean in salt water

• Further fermentation & sun-

7 drying the soybean
 • Boiling & washing black soybeans
1

Soy Sauce (Kecap) 2

• Sun drying the bean (half dry)

• Natural fermentation by keeping the

4 beans in the humid room (2-3 minggu)

• Keeping for 7d in salted water & boiling

5 the fermented soybean

• Separating the liquid from the dregs

6

• Mixing and cooking the liquid with palm

7 sugar

• Filtering the mixture

8
(Video from Trans7Official_YouTube)
• Mixing with other ingredient, keep it for
9 7d
Phenolic transformation
during fermentation:
Phenolic compounds
Phenolic compounds
OH
Phenol

Garcia-Salas, P., et. al, 2010

Main phenolic
compounds in
legumes
Isoflavonoids of Soybean

acetyl-

malonyl-

glucosyl-
 High level of consumption of soybean product linked to
low breast cancer risk of Japanese
Isoflavonoids

• Isoflavone bind to human estrogen receptors (hERs); resulting in estrogenic or antiestrogenic

activity; phytoestrogens; potentially benefit human health
• Protective against hormone dependent cancers (such as breast and prostate cancer);
Decrease menopausal symptoms
Phenolic transformation
during fermentation:
Methods: extraction, (tentative) structure
determination, quantification
 Methods: Extraction

Treated/untreated
Freeze drying
soybean

Extraction
with 70%
ethanol

UPLC-UV-MS/MS
Identification and
quantification
Structure determination: UV and MS Chromatogram
UV spectrum distinguish pterocarpans from others subclasses

260 226 227

343
225

324 284

0 0 0

fungus-
treated

4 6 8 10 12 14 16 18
Time (min)
RDA fragment give the information
prenyl position
luteone

2,3-dehydrokievitone

licoisoflavone A
RDA fragment give the information
prenyl position
luteone

2,3-dehydrokievitone

licoisoflavone A
RDA fragment give the information
prenyl position
luteone

2,3-dehydrokievitone

licoisoflavone A
C6 or C8? Comparing the relative intensities of diagnostic fragment ions
 Quantification of Phenolic Compounds
• Only few standard compounds were commercially available, direct quantitative analysis was
in most cases impossible
• An alternative would be to use a single reference compound to make a calibration curve,
calculate the amounts of other compounds of interest by extrapolation of their peak areas,
and account for the ratio of the molar extinction coefficient of the reference and that of the
compound of interest
• As only few molar extinction coefficients have been published. It was decided to keep the
quantification of crude extracts as transparent as possible, and use the aglycone of a main
phenolic as the calibrant, i.e. daidzein (for soybean and Phaseoleae), genistein (for lupine)
or trans-resveratrol (for peanut)
• The quantification was performed at one wavelength, representing the maximum
absorbance of the compound in question. The quantity of compounds in the crude extract
was expressed as milligram of external standard per gram dry weight, without correcting for
the difference in molecular weight between the compound of interest and reference
compound
Schematic illustration summarizing
the influence of light on the position
of prenylation of glycinol in fungus-
elicited soybeans. The block arrows
with black solid fill and white solid fill
represent the flux of prenylated
pterocarpan production obtained in
dark and light, respectively. The size
of the arrow indicates the importance
of the flux. Dashed arrows indicate
proposed conversions, awaiting
further proof.
Phenolic transformation
during fermentation:
Results
Food Fermentation: Modification of Food
Materials

Sharma, Ranjana, et. al, 2020

Cho, K.M. et. al, 2011
 OH

HO O
OH

O
OH OH
O

OH
Catechin gallate OH

Cho, K.M. et. al, 2011

• The role of a number of carbohydrate-
cleaving enzymes in phenolic antioxidant
mobilization from whole soybean during
solid-state fermentation
• Total soluble phenolic content increased
120 – 135% in the extracts
• Higher antioxidant activity was linked to
increased glucosidase and glucuronidase
activities
• High total phenolic content partly linked
to increased amylase activity
• The overall results (enzymatic activities
and phenolic antioxidant contents)
suggest the possible involvement of
lignin remobilization and/or degradation
activities, as well as phenolic
detoxification activities, by Rhizopus
oligosporus in phenolic antioxidant
mobilization from fermented whole
soybean.
McCue, P., Shetty, K. 2014
Isoflavonoids of edible
legumes:
Induction of (new) Phenolic Compounds
Phytoestrogens as food supplement or
therapeutic agents
• 1st generation phytoestrogens
HO O

OH O
OH
genistein

• 2nd generation phytoestrogens

HO O O
O O

O
O OH
OH
pterocarpan coumestan
Isoflavonoids in legumes are inducible

Fungal/microbial elicitors
Chemicals
Mechanical: wounding
Light

Defense mechanisme Defence molecules (Phytoalexins)

Isoflavonoids; prenylated
Plant-fungal
interaction

1. Mechanical barriers (a thick cuticle)

2. Chemical weapon:
• exudate compounds which inhibit spore germination and germ tube elongation
• the arsenal of constitutive (or preformed) antifungal compounds produced by plants
(phytoanticipins)
3. Blocking or delaying the advancement of the invader by producing reactive
oxygen species (ROS) as warning signals , triggering:
• Structural reinforcement of the cell wall
• The hypersensitive response (a programmed cell death)
• Development of systemic acquired resistance (SAR)
• The accumulation of newly produced antifungal chemicals (phytoalexins)
Grayer, R. J. (2000)
Challenging soybeans seeds by Rhizopus oryzae

Combined Malting and Fungus-Challenged

soaking germination fungus inoculation (7d)

(1d) (2d)
 Content of isoflavonoids increased, new
compounds induced
* * 1.30 mg DE/g DW

* *
Relative abundance

*
** *

2.95 mg DE/g DW
*
*
*
* *
* *
*
** * * *
* **
Different fungal elicitor to challenge germinated peanut

Arachis hypogea
 Aspergillus Vs Rhizopus on Peanuts
Prenylated stilbene
Germinated
Relative Absorbance 100

50

0
Rhizopus-elicited
100
Stilbenoid
50 dimers

0
Aspergillus elicited
100

50

0
4 6 8 10 12 14 16 18 20
Time (min)
 Fungal metabolism experiment

• + prenylated
stilbenoid extract
• Incubate (3d)

• Add acetone
• Filtration
Growing fungi in liquid • Evaporation
medium (4d) • Freeze drying
Prenylated stilbenoid
structures found in
peanut seedlings
elicited by fungi. The
stilbenoid phytoalexins
(in yellow area),
referred to as
unmodified prenylated
stilbenoids in the text,
were metabolized by
Rhizopus (solid arrows)
or Aspergillus (dashed
arrows) resulting in
modified prenylated
stilbenoids (in grey
area). The exact
position of
glycosylation, sulfation,
and hydroxylation of
these modified
prenylated stilbenoids
were not determined.
The fungus strikes back!

Metabolized /modified

Defence molecules