Utilization of Pumpkin Seeds (Cucurbita Moshcata D.)

in the Making of Fermented Drink

Eveline

and Paloma

Lecturer, Food Technology Department, Faculty of Sains and Technology, University of Pelita Harapan,

Jl. MH. Thamrin Boulevard 1100 Lippo Village, Kelapa Dua, Karawaci, Tangerang, Indonesia

Alumnus, Food Technology Department, Faculty of Sains and Technology, University of Pelita Harapan,

Jl. MH. Thamrin Boulevard 1100 Lippo Village, Kelapa Dua, Karawaci, Tangerang, Indonesia

Keywords: fermented_beverage, Lactobacilus_plantarum, pumpkin_seeds, skim_milk, Streptococcus_thermophilus

Abstract: Pumpkin (Cucurbita moschata Duchesne) is a type of Indonesian plants whose flesh is widely used as

traditional processed foods (dodol, kolak, chips), while the utilization of the seeds is not optimal. Pumpkin

seeds have been reported to contain bioactive compounds and functional nutrients (vitamins, fiber,

antioxidants) that can be increased by the fermentation process. This study aims to produce fermented

pumpkin seeds. Initially, the ratio of pumpkin seeds to water (1:3, 1:4, 1:5 [w/v]) and skim milk concentration

(5%, 10%) was determined on LAB fermentation of Lactobacillus plantarum : Streptococcus thermophilus

1:1. Analysis of pH, total of titrated acids (TTA), and total LAB showed a ratio of 1:3 and 10% skim milk to

provide products that meet the standards, which in a sequence are 4.34, 0.84%, 1.5×10

cfu/ml. The selected

formulation was then used to determine the ratio of L. plantarum and S. thermophilus (1:1, 1:2, 2:1). The

ratio of 1:1 is selected as the ratio that yields pH, TTA, and total LAB according to the standard, which in a

sequence are 4.27, 0.84%, 2.2×10

cfu/ml. The products with preferred formulation contains proximate

according to the standard (83.62% water, 0.84% ash, 2.30% milk fat, 4.00% protein, 9.24% carbohydrate [by

difference]), phenolic 643.27 mg GAE/l, flavonoid 612.00 mg GAE/l; as well as increased antioxidant activity

by 55.81% after fermentation (IC

291694.47 mg/l). The product is considered acceptable by panelists in

hedonic (4.05 of 7.0) with a good level of safety based on toxicity test of 786.90 ppm (low toxic) and free of

coiliform microorganisms.

1 INTRODUCTION

Pumpkin (Cucurbita moschata Duchesne) or more

commonly known by the name of “Parang”

Pumpkin/Calabash originated from America (Peru

and Mexico). This type of Indonesian plant is

included in the Cucurbitaceae family which is quite

potential, about 20-21 ton/hectare (Data Badan Pusat

Statistik dalam Kumala and Nurlaela, 2015).

Pumpkin is widely used as traditional processed

foods, such as dodol, kolak, and chips (Usmiati, et al.,

2005). The part of pumpkin that can be utilized in

addition to the fruit flesh is its seeds.

Research by Atuonwu and Akobundu (2010); El-

Aziz and El-Kalek (2011); Primawati (2007) states

that pumpkin seeds potentially have antioxidant

activity of 47.01%. Pumpkin seeds are a good source

of protein (39.25%) and is widely used as a diabetes

drug in mice in the African region (Teugwa, et al.,

2013), as well as worm medicines and medications

for functional bladder disorder in North America and

Mexico (El-Aziz and El-Kalek, 2011). Utilization of

pumpkin based on its functional potential, needs to be

increased again especially in the field of food such as

lactic acid fermented beverage containing lactic acid

and probiotics so as to provide health benefits for the

body (Vasudha and Mishra, 2013). According to

Wardani (2011); Mardianto, (2015); and Kencana

(2015), the fermentation process also increases the

antioxidant (flavonoids and phenolics) which add

value to fermented beverage products.

Ratio determination research of pumpkin seeds to

water. According to Ariyanto, et al. (2015), Primurdia

and Kusnadi (2014), and Usmiati and Utami (2008),

separately reveals the best ratio that can be used in the

manufacture of grain fermented beverages is 1:4 and

1:5; therefore in this study tested both these ratios and

also as a comparison is the ratio of 1:3. The addition

of skim milk is also a factor in the successful

Eveline, . and Paloma, .

Utilization of Pumpkin Seeds (Cucurbita Moshcata D.) in the Making of Fermented Drink.

DOI: 10.5220/0010040301870193

In Proceedings of the 3rd International Conference of Computer, Environment, Agriculture, Social Science, Health Science, Engineering and Technology (ICEST 2018), pages 187-193

ISBN: 978-989-758-496-1

187

manufacture of lactic acid fermented beverages.

Research done by Rachelia (2014) and Leo (2013)

found that 4% and 5% of skim milk can optimize the

fermentation process from peanut and tolo beans,

while Sari (2014) found 10% skim milk to make

fermented beetroot better. Therefore, in this study, it

is used 5% and 10% skim milk. The selected

formulation of the ratio and concentration of skim

milk was used in the next phase of the study (the

determination of culture ratio of Lactobacillus

plantarum and Streptococcus thermophilus) by pH

analysis, total titrated acid (TTA), and total lactic acid

bacteria (LAB) compared with the standard BSN

(2009), CODEX (2003), KEBS (2013), PNS (2007),

FSANZ (2014), and JETRO (2011).

Determination of treatment level of culture ratio

of L. plantarum and S. thermophilus based on

research done by Sari (2014) which found the best

amount of lactic acid and cell. This study used a ratio

level of 1:2, 1:1, and 2:1. The selected ratio was

determined through total LAB analysis, pH value,

total titrated acid also compared with the standard

BSN (2009), CODEX (2003), KEBS (2013), PNS

(2007), FSANZ (2014), and JETRO (2011). The

products with selected formulations are further

chemically tested (antioxidant, phenolic, flavonoid)

and microbiology (coliform), and evaluated by

proximate, toxicity, and hedonic test, so that this

pumpkin fermented beverage will meet the standard

of fermented beverages and may be accepted by the

panelists nutritionally functional and sensory.

2 MATERIALS AND METHODS

2.1 Materials

The main materials: pumpkin dried seeds, aquades,

skimmed milk powder, sugar, S. thermophilus, L.

plantarum. Man Rogosa Sharpe Broth (MRSB), Man

Rogosa Sharpe Agar (MRSA), Butterfield’s Phospate

Buffered, Lauryl Tryptose Broth (LTB), and Brilliant

Green Lactose Bile Broth (BGLBB). Analysis

materials: NaCl, 0.1 N NaOH, phenolphtalein

indicator, Folin-Ciocalteau reagent, sodium

carbonate, AlCl

, 25% HCl, DPPH, selenium, boric

acid, 98% H

35% NaOH, 0.02 N HCl, pH 4

buffer, pH 7 buffer, ethanol, dye solution, ascorbic

acid standard solution, HPO

-HOAc solution and

70% alcohol.

2.2 Methods

Phases of the research consisted of the phase of

making pumpkin seeds juice, phase 1 and phase 2.

Phases of pumpkin seeds juice extracts include

peeling skin from pumpkin seeds, seeds destruction

with blender, ratio of seeds:water = 1:3, 1:4, 1:5

(w/v), filtration and waste removal. Pumpkin seeds

juice according to each ratio was then used in phase 1

research. Phase 1 research (Figure 1) begins with the

addition of skim milk (5% and 10%) and sugar (5%)

in pumpkin seeds juice according to each ratio (1:3,

1:4, 1:5 [w/v]). Pasteurization was done (80°C, 15

minutes) and cooled to 45°C. Starter L. plantarum

(age 12 hours) and S. thermophilus (age 10 hours)

with a ratio of 1:1 were inoculated as much as 5% and

incubated at 42°C for 12 hours. Pumpkin seeds

fermented beverage was then analyzed for pH

(AOAC, 2005), total titrated acids (AOAC, 2005),

and total LAB (Wehr and Frank, 2004). The selected

formulation of the ratio of the pumpkin seeds to water

and the concentration of skim milk was used in the

next phase of the research through the comparison of

the results of each test parameter to standard BSN

(2009), CODEX (2003), KEBS (2013), PNS (2007),

FSANZ (2014), and JETRO (2011).

Phase 2 research (Figure 1) was done by

inoculating Starter L. plantarum (age 12 hours) and S.

thermophilus (age 10 hours) with a ratio of 1:1, 1:2,

and 2:1. The incubation conditions in the

fermentation process and the test analytical

parameters were performed the same as in the

previous phase. The culture ratios results that meet

the standard BSN (2009), CODEX (2003), KEBS

(2013), PNS (2007), FSANZ (2014), and JETRO

(2011) determined as the selected ratio. The

fermented beverages of the selected formulation and

ratio were then tested chemically (antioxidantt

activity [Nahak and Sahu, 2011], phenolic [Conde, et

al., 1997], flavonoids [Lamien-Meda, et al., 2008]) as

well as microbiological tests (coliform [BSN, 2006]).

Furthermore, it was evaluated proximately (water,

ash, fat, protein, carbohydrate by difference [AOAC,

2005]), toxicity [Lisdawati, et al., 2006], and hedonic

(scale 1-7).

2.3 Experimental Design

The experimental design of the Phase 1 research was

Completely Randomized Design of two factors. The

first factor (the ratio of pumpkin seeds to water)

contains three levels (1:3 [A1], 1:4 [A2], 1:5 [A3])

with three repetitions. The second factor (skim milk

concentration) contains two levels (5% [B1] and 10%

ICEST 2018 - 3rd International Conference of Computer, Environment, Agriculture, Social Science, Health Science, Engineering and

Technology

188

[B2]) with three repetitions. In the Phase 2 research

culture ratios of L. plantarum and S. thermophilus

was Completely Randomized Design one factor that

contained three levels (1:2 [A1], 1:1 [A2], and 2:1

[A3]) with repetition three times.

Pumpkin seeds

↓

peeling skin from pumpkin seeds

↓

Seeds destruction with blender on

(Seeds: Water ratio 1:3, 1:4, dan 1:5 (w/v))*

↓

Filtration

↓ →Waste (Remove)

Pumpkin seeds juice

↓

Add skim milk (5 and 10%) *and sugar (5%)

↓

Pasteurization in 80℃ for 15 minutes

↓

Cool down to 45℃

↓

Inoculate cultur L. plantarum dan S. thermophilus 5%

(1:1, 1:2, 2:1) **

↓

Incubate at 42℃ for 12 hours

↓

Pumpkin seeds fermented beverage

↓

Analysis

↓

TTA

↓

LAB value

↓

determination of the best formulation

↓

Analysis

Coliform

2. Toxicity

3. Proximate

4. Antioxidant activity

5. Phenolic content

6. Flavonoids content

7. Hedonic

↓

Pumpkin seeds fermented beverage

Note:

* = Phase I

** = Phase II

Figure 1. Research Flowchart

3 RESULT

3.1 Phase 1

The first phase research was conducted to determine

the best ratio of pumpkin seeds to water (1:3, 1:4. 1:5)

and skim milk concentration (5% and 10%) through

pH value analysis, total titrated acid value, and total

LAB based on comparison with standard BSN

(2009), CODEX (2003), KEBS (2013), PNS (2007),

FSANZ (2014), and JETRO (2011). L. plantarum and

S. thermophilus cultures were 5% inoculated at

optimum age of 12 hours (1.6×10

cfu/ml) and

10 hours (1.8×10

cfu/ml), respectively.

The result of statistical test between ratio of

pumpkin seeds and skim milk concentration showed

that both interact (p<0.05) influenced the pH value of

pumpkin seeds fermented. A number of nutrients in

the pumpkin seeds and milk lactose decompose as the

fermentation process becomes lactic acid and other

organic acids are formed (Wood, 1997; Retnowati

and Kusnadi 2014), thus lowering the pH value from

6.65 (control) to 4.09-4.49 (Table 1). The pH value of

all samples were still within the maximum standard

of 4.5 (FSANZ, 2014).

Results of the statistical test ratio of pumpkin

seeds and the concentration of skim milk showed no

interaction (p>0.05) influenced the TTA value of

pumpkin seeds fermented beverage, but each had

significant effect (p<0.05). Table 2 shows the lactic

acid and other organic acids are increasing with the

concentration of the pumpkin seeds juice and the

increasing concentration of skim milk. Energy

sources of pumpkin seeds and skim milk nutrients

increase the formation of lactic acid and other organic

acids during fermentation (Dewi, et al., 2013;

Mulyani, et al., 2013; Retnowati and Kusnadi, 2014;

Yanuar and Sutrisno 2015). During fermentation, the

TTA control value (0.33%) increased to 0.58-0.84%,

the value is still included in the standard of BSN

(2009) 0.2-0.9%; the sample ratio of 1:5 does not

meet the standards of PNS (2007) and CODEX

(2003) which more than 0.6%; and only 1:3 ratio and

skim milk 10% that meets KEBS standard (2013) 0.7-

0.9%.

Results of statistical test between ratio of pumpkin

seeds and skim milk concentration showed no

interaction to total LAB (p>0.05). The pumpkin juice

ratio did not affect the total LAB (p>0.05), whereas

the concentration of skim milk affected the total LAB

(p<0.05). Table 2 shows the total log value of LAB

during fermentation having increased from log 6.68

to ~log 8-9 both in the seeds ratio sample and on the

skim milk concentration sample. According to

Utilization of Pumpkin Seeds (Cucurbita Moshcata D.) in the Making of Fermented Drink

189

Vasudha and Mishra (2013), the fermentation process

will increase total lactic acid bacteria. Li, et al. (2012)

added that fermentation for 18-24 hours with more

than one culture can result in total lactic acid bacteria

of about 10

-10

cfu/ml (log 8-9). All test samples

meet the standard minimum of 10

cfu/ml (CODEX,

2003; PNS, 2007) and minimum 10

cfu/ml (BSN,

2009; FSANZ, 2015; JETRO, 2011).

Based on the analysis of pH value, TTA, and total

LAB compared with BSN (2009), CODEX (2003),

KEBS (2013), PNS (2007), FSANZ (2014), and

JETRO (2011), then the best formulation of pumpkin

seeds fermented beverages is the ratio of pumpkin

seeds to water and skim milk 1:3 and 10%. This

formulation has the results of analysis of each

parameter that meets all standards that is, pH 4.34,

TTA 0.84%, total LAB 1.5×10

cfu/ml; and will used

for phase 2 research in determining the best culture

ratio between L. plantarum and S.

thermophilus.

Table 1. Phase 1 Test Results (pH)

Pumpkin Seeds

Water

Skim Milk

(%)

1:3

5 4.09±0.02

10 4.34±0.03

1:4

5 4.13±0.01

10 4.36±0.02

1:5

5 4.37±0.03

10 4.49±0.00

Note: - Different notation showed there was significant

difference (p<0.05)

Table 2. Phase 2 Test Results (TTA and Total LAB)

TTA

(%)

Total LAB

(Log)

Pum

kin S

ed : Wate

1:3 0.84±

.08

9.12±0.05

0.69±0.11

9.01±0.11

1:5 0.58±0.15

9.05±0.0

Skim Milk (%)

5 0.62±0.15

8.99±0.05

0.78±0.1

9.15±0.0

Note: - Different notation showed there was significant

difference (p<0.05)

- No comparison between parameter analysis

3.2 Phase 2

Phase 2 research was conducted to determine the best

culture ratio between L. plantarum and S.

thermophilus through analysis of pH value, TTA, and

total LAB based on comparison with standard BSN

(2009), CODEX (2003), KEBS (2013), PNS (2007),

FSANZ (2014), and JETRO (2011). The results of

statistical test of culture ratio L. plantarum and S.

thermophilus showed an influence on TTA pumpkin

seeds fermented beverage (p<0.05). Table 2 shows

that the 1:1 ratio yields the highest TTA value

(0.84%) and meets all TTA value standards that is,

BSN (2009) 0.2-0.9%; CODEX (2003) and PNS

(2007) more than 0.6%; KEBS (2013) 0.7-0.9%. The

ratio of 1:2 and 2:1 both have no significant effect on

TTA value (0.60% and 0.66%) and significantly

affect the 1:1 ratio. According to Chandan and Kilara

(2013), the amount of Lactobacillus and

Streptococcus should be balanced so that

Lactobacillus can constantly provide peptide supply

decomposition result of Lactobacillus to

Streptococcus.

Results of statistical test of L. plantarum and S.

thermophilus showed that there was no effect on pH

value (p>0.05). Table 3 shows the entire sample

yielding pH values

 4.27-4.3 and meeting the pH

standard FSANZ (2015) of 4.5. According to Zhang,

et al. (2011), pH of skim milk which tend to be neutral

and can serve as buffer can cause pH value of three

culture ratios not significantly different. In addition,

the amount of H

ions only shows the acid content

dissociated in the sample and does not measure all the

acid content contained in the product such as total

titrated acids, so it can happen that the pH value is

significantly different but the TTA value is not

(Primurdia and Kusnadi, 2014).

The result of statistic test of culture ratio of L.

plantarum and S. thermophilus also did not show any

influence to total LAB (p>0.05). Table 2 shows the

three culture ratios result total LAB in the range of

log 9.28-9.34 that include in standard of CODEX

(2003) and PNS (2007), that is more than 10

cfu/ml.

Based on the analysis of pH, TTA, and total LAB

compared with BSN (2009), CODEX (2003), KEBS

(2013), PNS (2007), FSANZ (2014), and JETRO

(2011), then the best ratio of L. plantarum and S.

thermophilus as culture of pumpkin seeds fermented

beverage is a 1:1 ratio. This ratio has the results of

analysis of each parameter that meets all standards,

that is pH 4.27, TTA 0.84% (significantly different

from 1:2 and 2:1 ratio), total LAB 2.2×10

cfu/ml.

Pumpkin seeds fermented beverage with the selected

formulation of seeds ratio and the concentration of

ICEST 2018 - 3rd International Conference of Computer, Environment, Agriculture, Social Science, Health Science, Engineering and

Technology

190

skim milk (1:3; 10%) and the selected culture ratio

(1:1) were then chemically analyzed (antioxidant,

phenolic, flavonoid activity), microbiologically

(coliform), proximate, toxicity, and hedonic.

Table 3. Phase 2 Test Result (pH, TTA, and Total LAB)

Culture

Ratio

(ST : LP)

TTA

(%)

Total LAB

(Log)

1:1

4.27±0.02

0.84±0.04

9.34 ± 0.02

1:2

4.30±0.05

0.60±0.03

9.30 ± 0.04

2:1

4.30±0.05

0.66±0.04

9.30 ± 0.04

Note: - Different notation showed there was significant

difference (p<0.05)

- No comparison between parameter analysis

Based on the analysis of pH, TTA, and total LAB

compared with BSN (2009), CODEX (2003), KEBS

(2013), PNS (2007), FSANZ (2014), and JETRO

(2011), then the best ratio of L. plantarum and S.

thermophilus as culture of pumpkin seeds fermented

beverage is a 1:1 ratio. This ratio has the results of

analysis of each parameter that meets all standards,

that is pH 4.27, TTA 0.84% (significantly different

from 1:2 and 2:1 ratio), total LAB 2.2×10

cfu/ml.

Pumpkin seeds fermented beverage with the selected

formulation of seeds ratio and the concentration of

skim milk (1:3; 10%) and the selected culture ratio

(1:1) were then chemically analyzed (antioxidant,

phenolic, flavonoid activity), microbiologically

(coliform), proximate, toxicity, and hedonic.

The antioxidant activity test is performed to find

out how much antioxidant compound in the pumpkin

seeds fermented beverage can prevent radical

formation, inhibit oxidation reaction, decrease

oxygen concentration, metal chelating agent, and act

as bond breaker to prevent hydrogen removal from

the substrate (Winarsi, 2007). The analysis was

performed by DPPH method which measures the

sample ability in binding to free radicals expressed in

50.

The lower the IC

value, the higher the

antioxidant activity which means is a few amount of

sample is needed to bind 50% of the free radicals.

(Somawathi, et al., 2014). According to Putri and

Hidajati (2015), antioxidant activity has five

categories: very strong (IC

<50 mg/l), strong (IC

50-100 mg/l), moderate (IC

100-250 mg/l), weak

(IC

250-500 mg/l), and very weak (IC

>500 mg/l).

The results of antioxidant activity test (IC

)

pumpkin

seeds fermented beverage before and after

fermentation were 660142.2 mg/l and 291694.7 mg/l;

although relatively weak, antioxidant activity

increased due to increased lactic acid levels during

fermentation. In addition, probiotic bacteria can

produce vitamin C and E which can act as an

antioxidant (Kusumaningrum, 2011).

Tests of total phenolic compounds were

performed using the Folin-Ciocalcetau colorimetric

method (standard phenolic curve), while for total

flavonoids using the aluminum chloride colorimetric

method (standard flavonoid curve). The total test

results of phenolic and flavonoid pumpkin seeds

fermented beverage sequentially were 643.27 mg

GAE/l and 612 mg QE/l. The total phenolic and

flavonoid contained are interconnected with the

antioxidant activity obtained (Primurdia and Kusnadi,

2014).

Testing of coliform contamination (Gram

negative, not forming spores, rod shape, producing

acid and gas) is stated negatively on pumpkin seeds

fermented beverage, while the amount of coliform

allowed in fermented beverages is a maximum of 10

APM/ml (Badan Standarisasi Nasional, 2009). The

organic acid produced by LAB inhibits the growth of

coliform bacteria (Soccol, et al., 2013).

Proximate test in the form of moisture content, ash

content, fat, protein, and carbohydrate (by difference)

can be seen in Table 3. All test parameters include in

the standard of SNI except fat content but still fulfill

requirement of CODEX (2003) that is <15%.

Product toxicity test is performed as a preliminary

test in more complex toxicity testing. The Brine

Shrimp Lethality Test (BSLT) method is used to

produce LC

(Lethal Concentration 50) which is the

number of products needed to kill 50% of shrimp

larvae. According to Onzago, et al. (2014), LC

100 ppm: strong toxic, LC

100-500 ppm: moderate

toxic, LC

500-1000 ppm: low toxic, and LC

>1000

ppm: non toxic. The pumpkin seeds fermented

beverage has LC

786,90 ppm (low toxic).

According to Prasetia and Intan (2013), LC

<1000

ppm can be caused by components such as flavonoids

contained in the sample so that the product is still safe

for consumption.

The hedonic test of pumpkin seeds fermented

beverage was conducted on 70 panelists to determine

the level of product acceptance. The test scale

consists of 7: extremely dislike (1), dislike (2),

moderately dislike (3), neutral (4), moderately like

(5), like (6), extremely like (7). Average yields of

each parameters results is 3.29, taste 4.01, texture 4.4,

and overall 4.05. Overall the panelists evaluate the

product in the neutral category (4.05), that means the

product can still be accepted by the panelists.

Utilization of Pumpkin Seeds (Cucurbita Moshcata D.) in the Making of Fermented Drink

191

Table 4. Phase 2 Test Result (Proximate)

Parameters

Pumpkin

seeds

fermented

everage

SNI

Standard

(BSN,

2009)

CODEX

Standard

(CODEX,

2003)

Water (%) 83.62 - -

Ash (%) 0.84 max. 1.00 -

Fat

(

)

2.30 0.50-0.60 max. 15%

Protein

(

)

4.00 min. 1.00 min. 2.7

Carb. [b

diff]

(

)

9.24 - -

4 CONCLUSIONS

Preparation of pumpkin seeds fermented beverage

with a ratio of pumpkin seeds to water of 1:3,

addition of skim milk 10%, and culture ratio of L.

plantarum : S. thermophilus 1:1 result pH, TTA, and

total LAB that meet the standard. The pH, TTA, and

total LAB are respectively 4.27; 0.84%; 2.2×10

cfu/ml.

The products of this selected culture formulation

and ratio contains 83.62% water, 0.84% ash, 2.30%

fat, 4.00% protein, 9.24% carbohydrate, total

phenolic 643.27 mg GAE/l sampel, and total

flavonoids 612 mg QE/l. The product increased the

antioxidant activity by 55.81% after the fermentation

process, with IC

value after fermentation were

291694.7 mg/l. In hedonic, fermented beverage of

pumpkin seeds are still acceptable to consumers (4.05

of 7.00), and declared safe for consumption with low

toxicity and coliform-free microorganisms.

REFERENCES

Association of Official Analytical Chemist (AOAC). 2005.

“Official Methods of Analysis of AOAC International.”

AOAC International, Madison.

Ariyanto, N. O., Wiyanto, S. D., Hindarso, H., and

Aylianawati. 2015. Pengaruh rasio massa biji dan

volume air dan suhu ekstraksi terhadap ekstraksi biji-

bijian dalam pembuatan susu nabati. Jurnal Ilmiah

Widya Teknik 14(1): 20-25.

Atuonwu, A. C and Akobundu, E. N. T. 2010. Nutritional

and sensory quality of cookies supplemented with

defatted pumpkin (Cucurbita pepo) seed flour. Pakistan

Journal of Nutrition, 9: 672-677.

Badan Standarisasi Nasional (BSN). 2006. “SNI 01-

2332.1:2006: Cara uji mikrobiologi – Bagian 1:

Penentuan coliform dan Escherichia coli pada produk

perikanan.” Badan Standarisasi Nasional, Jakarta.

Badan Standardisasi Nasional (BSN). 2009. “SNI

7552:2009-Minuman susu fermentasi berperisa.”

Badan Standarisasi Nasional, Jakarta.

Chandan, R. C and Kilara, A. 2013. “Manufacturing Yogurt

and Fermented Milks”, Second Edition. John Wiley &

Sons, Inc, Chichester.

Codex Alimentarius Commission. 2003. “Codex Standard

for Fermented Milks.” Codex Alimentarius

Commission, Rome.

Conde, E. E., Cadahia, M. C., Garcia-Vallejo, B., Simon,

and Adrados, J. 1997. Low molecular weight

polyphenol in cork of Quercus suber. Journal of

Agriculture Food Chemistry: 2695-2700.

Dewi, E. C., Wulandari, S., and Sayuti, I. 2013. “Efektivitas

penambahan madu dan susu skim terhadap kadar asam

laktat dan pH yoghurt kacang hijau (Phaseolus

radiatus L.) dengan menggunakan inokulum

Streptococcus thermophilus dan Lactobacillus

bulgaricus.” Skripsi. Universitas Riau, Pekanbaru.

El-Aziz, A. B and El-Kalek, H. H. 2011. Antimicrobial

proteins and oil seeds from pumpkin (Cucurbita

moschata). Nature and Science 9(3): 105-119.

Food Standards Australia New Zealand (FRANZ). 2015.

“Standard 2.5.3: Fermented milk products.” Available

from

https://www.legislation.gov.au/Details/F2015L00413.

Accessed 1 Oktober 2016.

Japan External Trade Organization (JETRO). 2011.

“Specifications and Standards for Foods, Food

Additives, etc. Under the Food Sanitation Act

(Abstract) 2010.” Available from

https://www.jetro.go.jp/ext_images/en/reports/regulati

ons/pdf/foodext2010e.pdf. Accessed 1 Oktober 2016.

Kencana, A. H. 2015. “Aplikasi minuman fermentasi kulit

melinjo sebagai anti asam urat pada tikus wistar.”

Skripsi. Universitas Pelita Harapan, Tangerang.

Kenya Bureau of Standards (KEBS). 2013. “DKS 05-

941:2013-Specification for fermented (cutured) milks.”

Kenya Bureau of Standards, Nairobi.

Kumala, I and Nurlaela, L. 2015. Pengaruh penambahan

puree labu kuning dan lama pengocokan (agitasi)

terhadap sifat organoleptik es krim yoghurt. Boga 4(1):

202-210.

Kusumaningrum, A. P. 2011. “Kajian total bakteri

probiotik dan aktivitas antioksidan yoghurt tempe

dengan variasi substrat.”

Skripsi. Universitas Sebelas

Maret, Surakarta.

Lamien-Meda, A., Lamien, C. E., Compaore, M. M. Y.,

Meda, R. N. T., Kiendrebeogo, M., Zeba, B., Millogo,

J. F., and Nacoulma, O. G. 2008. Polyphenol content

and antioxidant activity of fourteen wild edible fruits

from Burkina Faso. Molecules 13: 581-594.

Leo, D. J. 2013. “Kajian minuman fermentasi sari kacang

tolo (Vigna unguiculata (L.) Walp) terhadap mikroflora

usus mencit”. Skripsi, Universitas Pelita Harapan,

Karawaci.

Li, S., Walsh, H., Gokavi, S., and Guo, M. 2012.

Interactions between Lactobacillus acidophilus strains

and the starter cultures, Lactobacillus bulgaricus and

Streptococcus thermophilus during fermentation of

goats milk. African Journal of Biotechnology 11:

11271-11279.

ICEST 2018 - 3rd International Conference of Computer, Environment, Agriculture, Social Science, Health Science, Engineering and

Technology

192

Lisdawati, V., Sumali, W., and Kardono, L. B. S. 2006.

Brine Shrimp Lethality Test (BLST) dari berbagai

fraksi ekstrak daging buah dan kulit biji mahkota dewa

(Phaleria macrocarpa). Buletin Penelitian Kesehatan

34(3): 111-118.

Mardianto. 2015. “Peranan minuman fermentasi daun

sirsak (Annona muricata L.) sebagai antikolesterol pada

tikus Sprague Dawley.” Skripsi. Universitas Pelita

Harapan, Karawaci.

Mulyani, S., Sudaryati, and Susanto, A. 2013. Kajian peran

susu skim dan bakteri asam laktat pada minuman

sinbiotik umbi bengkuang. Jurnal Teknologi Hasil

Pertanian 46-54.

Nahak, G and Sahu, R. K. 2011. Evaluation of antioxidant

activity in ethanolic extracts of five curcuma species.

International Research Journal of Pharmacy 2: 243-248.

Onzago, R.O., Kiama, S.G., Mbaria, J.M., Gakuya, D.W.,

and Nduhiu, J.G. 2014. “Evaluation of antimicrobial

activity and toxicity of vernonia hymenolepis (A.Rich)

traditionally used for toothache in Kenya.” Journal of

Phytopharmacology 3(1): 22-28.

Philippine National Standard (PNS). 2007. “PNS/BFAD

08:2007-Fermented milks-Specification.” Available

from

http://www.fda.gov.ph/attachments/article/153547/PN

S-BFAD%2008-2007-fermented%20milks-specs.pdf.

Accessed 1 Oktober 2016.

Prasetia, R and Intan, I. 2013. Uji toksisitas akut ekstrak

etanol buah lakum (Cayratia tryfolia) terhadap larva

Artemia salina Leach dengan metode brine shrimp

lethality test (BSLT). Prosiding Seminar Nasional

Kimia: 155-157.

Primawati, R. 2007. “Aktivitas antioksidan dan kadar

fenolik total biji semangka (Citrullus vulgaris schrad.)

dan biji labu kuning (Cucurbita moschata ex Poir).”

Skripsi. Universitas Kristen Satya Wacana, Salatiga.

Primurdia, E. G and Kusnadi, J. 2014. Aktivitas antioksidan

minuman probiotik sari kurma (Phoenix dactilyfera L.)

dengan isolat L. plantarum dan L. casei. Jurnal Pangan

dan Agroindustri 2(3): 98-109.

Putri, A. A. S and Hidajati, N. 2015. Uji aktivitas

antioksidan senyawa fenolik ekstrak metanol kulit

batang tumbuhan nyiri batu (Xylocarpus moluccensis).

UNESA Journal of Chemistry 4(1): 1-6.

Rachelia, M. Y. 2014. “Pemanfaatan bakteri asam laktat

dalam minuman fermentasi kacang tanah (Arachis

hypogaea L.)”. Skripsi, Universitas Pelita Harapan,

Karawaci.

Retnowati, P. A and Kusnadi, J. 2014. Pembuatan minuman

probiotik sari buah kurma (Phoenix dactilyfera)

dengan isolat Lactobacillus casei dan Lactobacillus

plantarum. Jurnal Pangan dan Agroindustri 2(2): 70-81.

Sari, Y. T. 2014 “Pemanfaatan sari bit merah (Beta

vulgaris L.) pada pembuatan minuman fermentasi

dengan bakteri asam laktat.” Skripsi. Universitas Pelita

Harapan, Tangerang.

Soccol, C. R., Pandey, A., and Larroche, C. 2013.

“Fermentation Processes Engineering in the Food

Industry”. CRC Press, Boca Raton.

Somawathi, K. M., Rizliya, V., Wijesinghe, D. G. N. D.,

and Madhujith, W. M. T. 2014. Antioxidant activity and

total phenolic content of different skin coloured brinjal

(Solanum melongena). Tropical Agricultural Research

26(1): 152-161.

Teugwa, C. M., Boudjeko, T., Tchinda, B. T., Mejiato P.

C., and Zofou, D. 2013. Anti-hyperglycaemic globulins

from selected Cucurbitaceae seeds used as antidiabetic

medicinal plants in Africa. BMC Complementary and

Alternative Medicine. 13:63.

Usmiati, S and Utami, T. 2008. Pengaruh bakteri probiotik

terhadap mutu sari kacang tanah fermentasi. J.

Pascapanen 5(2): 27-36.

Usmiati, S., Setyaningsih, D., Purwani, E. Y., Yuliani, S.,

and Maria, O. G. 2005. Karakteristik serbuk labu

kuning (Cucurbita moschata). Jurnal Teknologi dan

Industri Pangan 16 (2): 157-167.

Vasudha, S and Mishra, H. N. 2013. Non dairy probiotic

beverages. International Food Research Journal 20(1):

7-15.

Wardani, A. K. 2011. “Pengaruh fermentasi menggunakan

bakteri Lactobacillus bulgaricus terhadap kandungan

fenol total dan aktivitas antioksidan jus buah naga

merah (Hylocereus polyrhizus).” Skripsi, Universitas

Jember, Jember.

Winarsi, H. 2007. “Antioksidan Alami dan Radikal Bebas”.

Penerbit Kanisius, Yogyakarta.

Wehr, H. M and Frank, J. F. 2004. “Standard Methods for

Examination of Dairy Products”. American Public

Health Association, Washington.

Yanuar, S.E and Sutrisno, A. 2015. Minuman probiotik dari

air kelapa muda dengan starter bakteri asam laktat

Lactobacillus casei. Jurnal Pangan dan Agroindustri

3(3): 909-917.

Zhang, T., Zhang, C., Li, S., Zhang, Y., and Yang, Z. 2011.

Growth and exopolysaccharide production by

Streptococcus thermophilus ST1 in skim milk. Brazilian

Journal of Microbiology: 1470-1478.

Utilization of Pumpkin Seeds (Cucurbita Moshcata D.) in the Making of Fermented Drink

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