Development of Value-Added Products from Sago Frond

Ahmad, M.N.H.*; Matnin, N.; Adeni, D.S.A. and Bujang, K.B*.

Faculty of Resource Science and Technology

Corresponding Author: [email protected]

ABSTRACT

Sago frond is the compound leaf of the palm Metroxylon sagu. Our earlier studies revealed
that this material can be harnessed to produce Sago Frond Sugar (SFS), which contains
cellobiose and glucose as the main sugars at about 10g/L and 5g/L, respectively. SFS has
been proven as the comprehensive fermentation medium in production of L-lactic acid using
Lactococcus lactis IO-1. The yield and productivity L-lactic acid from SFS is comparable to
the Standard Medium (even when amended with yeast extract) at 0.85g/g and 85%,
respectively. SFS can be purified using Powdered Activated Charcoal (PAC) to produce
Purified Sago Frond Sugar (PSFS) and has been shown to have antibacterial properties
against several food related bacteria. Cellobiose from sago frond was perceived to be
beneficial as an antifungal agent when tested against Candida which is a common cause of
skin infection, where the growth of C. tropicalis is highly affected by SFS. However, this
purification process distinctly reduces its antifungal properties. In our attempt to increase the
sugar concentration (hence the enzymatic activities), SFS was heated to yield 50% and 100%
sugar concentrations. However, these too reduces its antifungal effect drastically by 20% and
0%, respectively. Sago frond sap, the liquid obtained by pressing freshly de-skinned sago
fronds on a roller press machine has been fermented to produce ethanol using the yeast
Saccharomyces cerevisiae. The sap contains higher amount of sugar (25 g/L) compared to
SFS (15 g/L). It was observed that sterilised SFS exhibited cell growth of up to 90% of the
yeast biomass. Astonishingly, similar growth was obtained when raw (unsterilized) sap was
used as the fermentation medium. This confirmed our conclusion that sago frond sap can be
used directly as the medium in large-scale ethanol fermentation without the need for
sterilisation. Recently, sago frond has also been utilised for the production of silage as animal
fodder. A combination of leaves and frond fibres can generate 42 tons of silage from the
harvesting of approximately 500 sago logs, every day. In our preliminary trials, the best
practice is to mix shredded sago leaves with sago frond sap which enhanced the lactate
fermentation using Lactococcus lactis IO-1 to produced nutritious silage. L. lactis IO-1 was
recycled as the inoculant to increase the fermentation efficiency and quality of sago frond
silage.

Keywords: sago fronds, sago leaves, cellobiose, lactic acid, ethanol, silage, animal feed.

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 INTRODUCTION

This is a review paper, covering previous works on production of lactic acid from sugars
obtained by enzymatic hydrolysis of pulverised sago frond fibres. The sugar, termed Sago
Frond Sugar (SFS) contains mostly cellobiose together with some glucose. Direct
fermentation of SFS without for further amendment has been achieved with considerable
success on the yield of L-lactic acid, an important commodity with various uses. From here,
our on-going research continues on the use of cellobiose due to its antibacterial and anti-
fungal properties. The latest development is the use of raw sago frond sap as the medium for
ethanol fermentation, generating high amount of biomass when it is sterilised and amended
with yeast extract.
It has been postulated that by the by the year 2050 food production needs to be amplified by
as much as 60% to meet global demand due to speedy expansion of the human population
(FAO, 2012). Unfortunately for some countries, fertile land is getting scarce and clean water
sources are diminishing. Ignoring this target will certainly affect our world in terms of food
security, stability and general peace (Konuma, 2018). Alternative source of food from the
hardy, underutilised and unpretentious sago palm that generates starch at 20-25 ton/ha
(Ishizaki, 1997), suddenly becomes very interesting. Since the average annual intake of starch
per person is approximately 250 kg, it has been postulated that a 1,000ha sago farm can
support and save 100,000 people from starvation (Ishizaki, 1997).
The largest sago palm growing areas (wild and semi-cultivated) is Indonesia, followed by
Papua New Guinea. But the largest systematically cultivated sago estate (approx. 50,000 ha)
supplying logs to modern factories that extracts, dries and packs totally white food-grade
sago starch in just over 30 minutes upon rasping is in Sarawak, Malaysia. Since the year
2000, Sarawak has established new sago plantations mainly in the districts of Mukah, Dalat,
Igan and Pusa. However, the long delay (10-12 years) prior to harvesting together with a
minimum of other saleable products from sago farms are two of the biggest challenges to
expand the sago industry.
This paper focuses on multiple uses of the sago fronds to augment the income of farmers
between and after harvesting of the sago palms. In the district of Mukah in Sarawak,
approximately 500 sago palms are harvested daily to feed the eight large factories, each one
generating between 20 to 30 tons starch/day. At this rate, and at about 10-15 fronds/palm, a
minimum of 5,000 fronds with a total weight of 30 tons is discarded into the environment
each day, unutilised (Ahmad, 2015). We have revealed that enzymatic hydrolysis of sago
frond pith can produce cellobiose, a potential substrate in production of lactic acid (Ahmad,
et al., 2016). Our current work focuses on sago frond sap which has been shown to be highly
fermentable by yeast to produce ethanol.

1. LACTIC ACID
Lactic acid is widely used in a host of industries as in food and beverages, cosmetics (skin
care, toiletries and hair care products), pharmaceutical and various industrial applications
including textile, chemical, metal and cleaning agents. However, only L-lactic acid can be
used in beauty products due to its complete absorption and compatibility with the human
skin. This can be produced by microbial fermentation using the lactic acid bacteria

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Lactococcus lactis IO-1 due to its ability to produce only L-lactic acid (Ishizaki and Okta,
1989; Jolhiri and Bujang, 1998; Bujang et al, 2000).
Sago frond contains large quantities of lignocellulose components (15.8% hemicellulose,
41.4% cellulose and 6% lignin). Enzymatic hydrolysis of dried sago frond powder using
cellulase enzyme produces Sago Frond Sugar (SFS), made of cellobiose (16-18%) and
glucose (9-11%). SFS also contain Mg (72.66 mg/L), Mn (18.37 mg/L) and Cu at 0.04 mg/L
which are needed by Lactic Acid Bacteria (Bujang et al., 2017).
The potential of SFS for production of lactic acid was analysed on three types of fermentation
media; Standard Medium (SM, made of commercial glucose and yeast extract at 4:1 ratio),
Sago Frond Sugar amended with yeast extract (SFS+YE) and only Sago Frond Sugar (SFS).
As expected, production of lactic acid was highest from the SM medium (22.81g/L) with
94% sugar consumption and 94% lactic acid recovery, obtained after 42 hours (Table 1).
Comparable yield was observed in SFS+YE medium at 21.40g/L and 96.2% for production
of lactic acid and sugar consumption, respectively. This was achieved 6 hours later, at 48
hours. However, it was observed that the SFS medium produced only slightly lower yield of
lactic acid at 19g/L within the same duration (48 hours), reflecting the advantage and
economics of using SFS as it is for future large-scale processes in reducing fermentation
costs.

Table 1: Production of Lactic Acid and sugar consumption from Standard Medium (SM), Sago Frond
Fermentation Media
Parameters SM SFS+YE SFS
Lactic acid (g/L) 22.81g/L 21.40g/L 18.64g/L
Residual sugars (g/L) 0.85 0.91 4.23
Sugar consumption (%) 93.8 96.2 84.8
LA recovery (%) 94.2 89.2 80.8
Sugar amended with Yeast Extract (SM+YE) and only Sago Frond Sugar (SFS).

As expected, slightly higher (5%) yield of lactic acid (0.90g/g) and other kinetic parameters
(productivity 0.54g/L/hr, efficiency 90.8%) was evident when using Standard Medium (SM)
compared to the other two media, being the complete medium with glucose as the main
carbon source (Table 2).

Table 2: Kinetic parameters in production of L-lactic acid from Standard Medium (SM), Sago Frond
Sugar amended with Yeast Extract (SFS+YE) and Sago Frond Sugar only (SFS).

Fermentation Media
Parameters SM SFS+YE SFS
Lactic Acid Yield (g/g) 0.90±0.005 0.85±0.006 0.85±0.001

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 Productivity (g/L/hour) 0.54±0.003 0.45±0.009 0.39±0.001
Efficiency (%) 90.81±0.53 85.03±0.63 84.76±0.85

Only slightly lower yield at 0.85g/g, productivity at 0.45g/L/hr at 85% efficiency was
observed in the medium SFS+YE. The obvious advantage is that, the yield and efficiency of
the process in only SFS medium is still the same at 0.85g/g and 85%, respectively. This
confirmed that SFS can be fermented into lactic acid as it is with comparable outcomes to the
Standard Medium (SM). The sap can be extracted easily from sago frond using a roller-press
machine. It contains free sugars, primarily glucose (43.8 g/L) and xylose (17.3 g/L), together
with residual starch (5.55 g/L). Enzymatic hydrolysis followed by sterilisation can maximise
sugar yield rendering it suitable as the substrate for L-lactic acid fermentation. The ability of
L. lactis IO-1 to metabolise both glucose and xylose promoted the sugar consumption up to
89.93% while amendment with salt and neutralizing agent allow L. lactis IO-1 to effectively
consume sugar higher than normal threshold of 30 g/L. This encourages growth of biomass to
the maximum which later can be used as the inoculant in silage manufacture (Ahmad et al,
2022).

2. HAND SANITISER
This hand sanitizer (registered as SaFrond) was developed from L-lactic acid produced from
fermentation of sago frond sugar. Selective antibacterial properties of SaFrond protect the
skin from pathogenic bacteria while other beneficial bacteria indigenous to the skin is not
affected, hence creating a balance of microflora on our skin. SaFrond hand sanitizer is totally
non-toxic due to the organic ingredient contents, hence completely harmless should it is
accidentally consumed. It uses organic fragrance (phthalates free) from pandan extract
(Pandanus amaryllifolius) and free from any preservatives. SaFrond hand sanitizer is
completely Halal due to the absence of alcoholic contents and thus attracts a larger portion of
consumers (Bujang et al., 2017). The main advantage of this project is that it minimises
disposal of sago fronds, both from pruning and harvesting. Once commercialised, this
product can generate extra income to the sago farmers.

3. ANTIFUNGAL CLEANSER
Purified Sago Frond Sugar (PSFS) produced by filtration on Powdered Activated Charcoal
(PAC) has been shown to have antibacterial properties against several food related bacteria
such as Staphylococcus aureus, Escherichia coli and Salmonella typhi (Ahmad and Bujang,
2014). Antimicrobial agent develops from organic or natural material is useful in order to
meet the demand of newer drug that is safe to use. It was reported that cellobiose derivative
has the highest antimicrobial activity against the bacterium E. coli (Antonio et al., 2010). In
this study, cellobiose was tested on Candida to observe its potential against fungal infection.
Preliminary analyses showed that both SFS and purified sago frond sugar (PSFS) affects only
C. tropicalis and not C. albicans, exhibited as the absence of inhibition zone on agar plates.
Meanwhile, SFS was highly effective against C. tropicalis with an average clearing zone of
9mm. This was somewhat reduced to 7mm when it was heated and concentrated to 50% in
our attempt to increase its antifungal properties. PSFS have lesser activity against C.
tropicalis at a clearing zone of lower than 6mm which disappears altogether when

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concentrated to 50%. High concentration increases the viscosity of the sago frond sugars and
reduced its penetration into the agar, hence decreasing antifungal activities.

4. ETHANOL
Ethanol has been studied before this using sugar from pure sago starch (Adeni and Bujang,
1998; Bujang et al, 2000) but starch being a source food, it is not ethical as a substrate for
large-scale production of energy.
A young sago frond (skinned) in the middle circle of the crown weighs about 5-6 kg, slightly
heavier than old ones. Around 1200ml of sap can be extracted from the whole frond, hence a
calculated ratio of about 1kg frond to 200ml sap. Since each sago palm harbour between 10-
15 fronds, a minimum of 12L sap/palm can be procured, or a total of 6,000L from an
estimated harvest of 500 palms/day in the Mukah District in Sarawak.
Due to our hot climate, the amount of sap is highly affected by the duration after harvesting
and a loss of about 25% is typical after 24 hours. The sap is greenish yellow in colour (not
dissimilar to sugar cane juice) and has an acidic pH between 5.0 to 5.1. It contains higher
amount of glucose (25.18 g/L) compared to sago frond fibre (15 g/L), an important criterion
in large-scale ethanol or lactate fermentation.

Preliminary trials on growth of yeast (Saccharomyces cerevisiae) on neat sago frond sap is
shown in Figure 1. On its own, only some increased (25%) in biomass was observed when
SFS was autoclaved, which does not justify the need for it to be sterilised.

Figure 1: Effects of sterilisation on biomass of yeast in raw sago frond sap.

However, addition of 5% w/v yeast extract (YE) generates a 90% difference in the amount of
biomass (as Dried Cell Weight, DCW) on raw SFS, after 24 hrs of growth (Figure 2).
Clearly, the sap does not have to be autoclaved in our trials, addition of YE is sufficient to
increase the biomass, and the subsequent production of ethanol.

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Figure 2: Effects of yeast extract on biomass of yeast in raw sago frond sap

However, when autoclaved, addition of yeast extract produced higher biomass at over 25%
(4.5 g/L, Figure 3) compared to 3.3 g/L when using raw or unsterilized SFS, as shown
previously. Due to the cost of sterilisation for large-scale processes, it is sufficient just to add
yeast extract to enhance cell growth. These preliminary trials clearly support the possibility of
producing ethanol directly from sago frond sap, without the need for sterilisation.

Figure 3: Effects of autoclaving and amendment with yeast extract on biomass of yeast

5. ANIMAL FEED
During the onset of this paper, the animal feed project was fairly new to our lab and we were
in the process of exploring the best practice, mixture and supplement to enhance edibility of
the silage produced. Silage from sago frond was prepared using leaves, preferably of the non-
thorny variety. The leaves were removed from the frond, cut into small pieces (2-3 cm) and
pulverised using a dry grinder. Silage from leaves is suitable for calves and young animal. In
order to enhance the protein and sugar content, fibres from the frond upon extraction of the
sap was added and pounded together. The mixture is promptly packed inside high-density
plastic bags and vacuumed to remove air, and kept at room temperature for at least one week.
Absence of oxygen enhances the fermentation process which causes drastic degradation of
protein in the silage together with excess in ammonia. This reduces the quality of the silage
and severely affects the animal leading to infertility, reduces lactation, lower rumen activity
and even death. Surplus production of ammonia can be avoided by enhancing the
acidification process during silage fermentation (Lallemand, 2018). Using Lactococcus lactis
IO-1 (from production of lactic acid on sago frond sap) indeed enhanced acidification during

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silage fermentation and concomitantly inhibits activity of the protease enzyme and the
production of ammonia. This inadvertently will maintain quality of the silage and ensure
safety of the livestock.
Clearly, numerous products which have the potential to be commercialised are available in
order to support our cash-strapped sago farmers while further developing our sago industry.
The common notion of sago farming that we have to wait for 10 to 12 years before we can
reap what we planted is long gone. Our current studies on the use of sago leaves to generate
silage and sugar from both sago starch (Bujang et al., 2000; Bujang, 2015) and sago fibre or
hampas (Adeni et al., 2013) are definitely achievable with lesser technologies, in tandem
with minimising impact on the environment from the sago industry.

ACKNOWLEDGEMENTS
The author sincerely acknowledged financial contributions from the Sago Chair of UNIMAS,
Fundamental Research Grant Scheme (FRGS 2017) and Special Fundamental Research Grant
Scheme (spFRGS 2017) from the Ministry of Science, Technology & Innovation, Malaysia.

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