Assessment of current hygienic manufacturing practices and evaluation of additional food preservatives effect in Nam Prik Mushroom

ASSESSMENT OF CURRENT HYGIENIC
MANUFACTURING PRACTICES AND EVALUTION OF
ADDITIONAL FOOD PRESERVATIVES EFFECT
IN NAM PRIK MUSHROOM

BERLIAN PURNAMA SARI

DEPARTMENT OF FOOD SCIENCE AND TECHNOLOGY
FACULTY OF AGRICULTURAL ENGINEERING AND TECHNOLOGY
BOGOR AGRICULTURAL UNIVERSITY
BOGOR
2015

STATEMENT LETTER OF MANUSCRIPT AND SOURCE
OF INFORMATION
I declare the truth that this manuscript entitled Assessment of Current
Hygienic Manufacturing Practices and Evaluation of Additional Food
Preservatives Effect in Nam Prik Mushroom is my own work with guidance of the
advisors and has not been submitted in any form at any college, except Bogor
Agricultural University and Prince of Songkla University. Sources of information
derived or quoted from published and unpublished works of other authors mentioned

in the text and listed in the list of references at the end of this manuscript.
Bogor, January 2015
Berlian Purnama Sari
NIM F24090118

i

ABSTRACT
BERLIAN PURNAMA SARI. Assessment of Current Hygienic Manufacturing
Practices and Evalution of Additional Food Preservatives Effect in Nam Prik
Mushroom. Supervised by DEDI FARDIAZ (Bogor Agricultural University);
BENCHAMAPORN PIMPA, SOMWANG SONGSAENG, PARAMEE NOONIM
(Prince of Songkla University, Suratthani Campus, Thailand).
Nam Prik Mushroom is ready-to-eat food product in Thailand which made
from edible mushroom Schizophyllum commune with curry paste. It is produced by
Chaiyo Farm, one of Thailand small-medium enterprise. Current hygienic and
manufacturing practices in Chaiyo Farm and effectiveness of combination chemical
food preservatives, propyl-paraben and calcium propionate, of Nam Prik Mushroom’s
shelf life was assessed.
Assessment of manufacturing practices at Chaiyo Farm

resulted in total conformity of 25.83% based on Thailand Primary Good
Manufacturing Practices. Propyl-paraben and calcium propionate did not influence
pH, color, and water activity of Nam Prik Mushroom. The effectiveness combination
of propyl-paraben and calcium propionate to extend the shelf life Nam Prik
Mushroom was not proven. Either non-preserved or preserved Nam Prik Mushroom
could not be accepted organically after 3 days storage at room temperature.
Keywords:
GMP, propyl-paraben, calcium propionate, Schizophyllum commune, shelf life

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ASSESSMENT OF CURRENT HYGIENIC
MANUFACTURING PRACTICES AND EVALUTION OF
ADDITIONAL FOOD PRESERVATIVES EFFECT
IN NAM PRIK MUSHROOM

BERLIAN PURNAMA SARI

Manuscript
In the partial fulfillment of requirement for degree of

Bachelor of Agricultural Technology
at
Departement of Food Science and Technology

DEPARTMENT OF FOOD SCIENCE AND TECHNOLOGY
FACULTY OF AGRICULTURAL ENGINEERING AND TECHNOLOGY
BOGOR AGRICULTURAL UNIVERSITY
BOGOR
2015

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PREFACE
Praise to The Lord for His mercy, bless, and love throughout the study in food
science and finished this manuscript for partial fulfillment of bachelor degree.
1. In particular, I want to appreciate Mama, Papi, Mama Yan, Mama Yanti,
Abang Yudha, Kokoh Opan, Cera, Julian, and all my family for their
continuing encouragement.

2. I would like to grateful thank and deeply indebted to Prof. Dr. Ir. Dedi
Fardiaz, MSc, my academic advisor in IPB; Assist. Prof. Dr. Benchamaporn
Pimpa, Dr. Somwang Songsaeng, Dr. Paramee Noonim, my research
advisors in Prince of Songkla University Suratthani-Thailand; for the
welcome, advices, encouragement, valuable times, and for my report
correction.
3. Grateful thank to Department of Food Science and Technology Bogor
Agricultural University-Prince of Songkla University Suratthani, who had
provide the student exchange program.
4. I am deeply thanks to Dr. Dra. Suliantari, MSi and Dr. Ir. Yadi Haryadi,
MSc; for giving their valuable times and help to be my final examiner.
5. I would like to thank to Prof. Dr. Ir. Ratih Dewanti-Hariyadi, MSc; for
giving support and advices.
6. I am heartfelt thank to P’Boo and the gank for helping me in my research
and the wonderful time together.
7. I am thanks to Ajarn Sutida, Ajarn Russel, P’ Pipe, and Ryan for caring and
supporting me during stayed in Thailand.
8. I am deeply thanks to Brian, who had accompanied me in Thailand and gave
me support every time.
9. Unforgetful, May, Tang, Pi, Dia, Tick, Mayae, Coy, Kim, Bird, and all my

PSU friends, big thanks for the unforgettable moment that we had.
10. I am very thanks to Anela, Doni, and “Pitung & Kumpeni” Ririd, Astro,
Sarlub, Nurhad for helping and supporting me until now. Also, thanks a lot
to all my lovely friends ITP46, Sri Rahayu I lovers, Kosan Bidadari, and
others.
Thank you very much!

Bogor, January 2015
Berlian Purnama Sari

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TABLE OF CONTENT
LIST OF TABLE

vi

LIST OF FIGURE

vi

LIST OF APPENDIX

vi

1. INTRODUCTION

1

1.1. Background

1

1.2. Research Objectives

2

2a. MATERIALS AND METHODS
Assessment of Current Hygienic and Manufacturing Practices
3a. RESULT AND DISCUSSION

Current Hygienic and Manufacturing Practices in Chaiyo Farm
4a. CONCLUSION & RECOMMENDATION

3
3
5
5
11

4a.1. Conclusion

11

4a.2. Recommendation

11

2b. MATERIALS AND METHODS

13

2b.1. Product Preservation Using Propyl-paraben and Calcium Propionate

13

2b.2. Analysis

13

2b.2.1. Sample Collection

13

2b.2.2. Physicochemical Analysis

14

2b.2.3. Microbiological Analysis

14

3b. RESULT AND DISCUSSION

17

Product Evaluation
4b. CONCLUSION & RECOMMENDATION

17
21

4b.1. Conclusion

21

4b.2. Recommendation

21

REFERENCES

23

APPENDIX

27

AUTHOR BIOGRAPHY

43

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LIST OF TABLE
1

Percentage Conformity and Non-conformity of Chaiyo Farm GMP based on
Thai Primary GMP

5

LIST OF FIGURE
1
2

Chaiyo Farm’s Nam Prik Hed
Schizophyllum commune

1
2

LIST OF APPENDIX
1
2

Nam Prik Hed making process
Assessment checklist and corrective actions of Nam Prik Hed
production plant
3 Chaiyo Farm’s production building and manufacturing practices
4 Effect of preservative on physicochemical Nam Prik Hed
5 Total aerobic plate and total yeast & molds of Nam Prik Hed
6 Total aerobic plate count in PCA mediun
7 Total yeast and mold in MEA medium
8 Microbiological quality of processing plant, laminar flow, and packaging
9 Personal observation results
10 Hazard analysis
11 Critical control point determination

27
28
34
35
35
36
36
37
38
39
41

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1. INTRODUCTION
1.1. Background
Nam Prik Mushroom is a ready-to-eat product that made from California fungi
with chilli or curry paste. People eat Nam Prik Mushroom together with rice.
Chaiyo Farm, one of Thailand small-medium enterprise, produces Nam Prik
Mushroom in type of flavor, curry and chili paste. Texture of this food is soft and
moist. After packaging, producer stores the product in a refrigerator. See Nam
Prik Mushroom of Chaiyo Farm on Figure 1.

Figure 1. Nam Prik Mushroom

Producer displays Nam Prik Mushroom in a booth located on roadside, in
front of Chaiyo Farm’s mushroom farm. One cup of Nam Prik Mushroom
weighed is about 50 grams. Shelf life of Nam Prik Mushroom that Chaiyo Farm
produced is only 3-4 days in refrigerator. This shelf life can be obtained when
washing raw mushroom was being a part of Nam Prik Mushroom making process
and product stored in a refrigerator.
California fungi (Schizophyllum commune) is shell-shaped (1-5 cm), thin,
small, usually in groups. On upper surface, it has grey-brown to flesh-colored
becoming white with dryness, downy-wolly. On lower surface, it appear as gilled,
hymenium covering fan-like arranged (See figure 2). Schizophyllum commune is
included in edible mushroom category. Initially, it was eaten in Assam, Congo,
Peru, and Thailand, and used as chewing gum in Hong Kong, Indonesia, and
Malaysia (Schimdt, 2006). In Chaiyo Farm, Schizophyllum commune is collected
from their own farm not far from the production area.
Good Manufacturing Practices (GMP’s) means all production steps which
include: procedures, start from location, building, sanitation facilities, production
equipment, material, processing, final product, laboratory, employee, packaging,
labeling, storage and maintenance to prevent food contamination from food safety
hazard (Indonesia Ditjen PPHP, 2009). It also control product quality standard in
line to their specification and produce the product consistently (WHO,1997).

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According to notification from the Thailand Ministry of Public Health

Figure 2. California Fungi
Thailand (NO 342) B.E. 2012, the requirements for implementation of GMP
consist of: 1) location and building; 2) tools, machinery, and production
equipments; 3) control of production process; 4) sanitation; 5) cleaning and
maintenance; 6) personnel and employee hygiene. Chaiyo Farm shall implement
GMP’s to increase traditional producer’s revenue. Prior to implementation,
preliminary assessment of current manufacturing practices in Chaiyo Farm shall
be carried out.
Propyl-paraben and calcium propionate are two food grade chemical
compounds that act as food preservative. These food preservatives possibly can
increase the shelf life of Nam Prik Mushroom because both of them are able to
work at neutral pH. Propyl-paraben is stable at a broad pH range and heat stable
during cooking process (Msagati, 2012). It is used at 100 to 1000 ppm (0.01 to
0.1%) in food (Ray,2005). Calcium propionate is able to work at higher pH
values. Gram-negative bacteria are inhibited by propionate. However, it is not
good to against yeast (Tucker, 2007). The maximum level to use it is 0.3% if it is
used alone (Smith & Hong-Shum, 2011).
1.2. Research Objectives
This research aimed to assess current hygienic and manufacturing
practices in the factory before GMP implemented in Chaiyo Farm. It was also
carried out to measure an effect of additional propyl-paraben and calcium
propionate in Nam Prik Mushroom.On the Insert tab, the galleries include items
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2a. MATERIALS AND METHODS
Assessment of current hygienic and manufacturing practices was carried
out in November 17 th 2013. Study location was in Chaiyo Farm, Suratthani,
Thailand.
Assessment of Current Hygienic and Manufacturing Practices
This step aimed to provide information on the current hygienic and
manufacturing practices adopted in the factory before the implementation of
GMP. Assessment was carried out by an assessment checklist (see Appendix 2).
Assessment checklist was prepared based on Primary GMP Thailand Guideline
(Thailand Ministry of Public Health, 2012). A total of 35 Good Manufacturing
Practices aspects based on the basic requirements was checked at the factory.
There were three checklist points in assessment checklist, such as conformity
(when the requirements were fully obeyed), non-conformity (when the
requirements were partially obeyed or not obeyed), and not applicable. Noapplicable points were not calculated in determining the percentage of conformity
and non-conformity (Dias et.al., 2012).
Assessment began while researcher was interviewing producer on her
perspective about hygiene, sanitation, and sequences of production process.
Afterwards, researcher checked building and production process which consists of
preparation, cooking, cooling, and packaging.

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3a. RESULT AND DISCUSSION
Current Hygienic and Manufacturing Practices in Chaiyo Farm
Chaiyo Farm has not fully implemented Primary GMP yet in processing
of their product. From the assessment results, Chaiyo Farm production building
reached 25.83% conformity based on Thailand basic requirements of Primary
GMP (Table 1). Non-conformity of Chaiyo Farm’s production building and
required corrective action are shown in Appendix 2. Limitation from this part was
researcher asked by producer and advisor’s assistant to participate in production
process. It aimed to accelerate Nam Prik Mushroom making process.
Table 1. Percentage Conformity and Non-conformity of Chaiyo Farm GMP based
on Thai Primary GMP.
Assessed Aspects
Conformity
Non(%)
Conformity
(%)
Location and production buildings (10 aspects)
10
90
Tools, machinery, and production equipment
100
0
(3 aspects)
Control of production process (4 aspects)
20
80
Sanitation (5 aspects)
0
100
Cleaning and maintenance (3 aspects)
0
100
Personnel and employee hygiene (8 aspects)
25
75
AVERAGE
25.83
74.17
3a.1. Location and production buildings
Conformity of Chaiyo Farm’s manufacturing practices of Location and
Production Buildings assessed aspects (10%) was adequately lighting and
ventilation in production area for supporting employee’s performance. As much as
90% from 10 points assessed aspects was not complied with Thai Primary GMP.
See appendix 3 to know Chaiyo Farm’s production building and manufacturing
practices.
Chaiyo Farm’s processing area was a kitchen of processor’s house and
connected to residential areas. Everyone and pets could enter production building.
There were birds, a cat, and a dog inside of production building in the production
area. Near the production area, researcher found unused materials, such as
washing machine, trash can, chairs, packaging materials, dish of pet, shoes rack,
and etc. Outside building’s floor was covered by any mosses. Building was right
beside a yard (soil). It also was near the road. It was exposed directly onto the
road. There was no full-wall to cover it. Drained pipeline connected to public
sewage pipelines was less good. Internal pipeline was not provided. The sanitation
program was carried out at once per day after product processing.
Contamination risk will increase if production building is connected to
residential area. Personnel or pet entered production area may cause cross
contamination or bring contaminants from outside to inside. Unused materials,

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mosses of the outside building’s floor, the yard, can be sources of contaminants.
Location near the road and the wall not covered fully led production area exposed
to high amount of dust. Inappropriate drained pipeline and unavailable internal
pipeline can cause a puddle around production building. Sanitation program
before production process will prevent any contamination to final product
(Winarno & Surono, 2004).
Producer shall move unused materials (bird cages, chairs, shoes racks,
food dogs, etc) from the processing area to other places. Processing building
should be move to other area that not exposed of dust too much. Producer can
build a new building or rent a house specially for product processing. A new
building should be built for production area particularly (separated from
residence). The sanitation program in the building shall be carried out before and
after product processing. The production area shall be covered by full wall with
adequate window, ventilation, and also give a door to enter the production area.
Internal pipeline shall be built and extended to public sewage system. Producer
also shall renovate drained pipeline (Thailand Ministry of Public Health, 2012).
Production lines and product flow should be designed to have an one
direction or linear flow from raw materials to finished product (for example: from
receiving, to storage, to preparation to, packaging). It aims to prevent cross
contamination (CFIS, 2013).
3a.2. Tool, machines, and production equipment
Tool, machines, and production equipment aspects of Chaiyo Farm were
about 100% complied Thai Primary GMP. Material of production equipments was
appropriated. It was made from non-hazardous material, such as stainless steel.
Tables and surface area were built by ceramics. It made easy to clean the
equipments. Tools and equipments design were suitable for cooking process of
Nam Prik Mushroom.
However, producer should change old equipment to the new one if frying
pan had uneven surface. Contamination may be occurred from food residue which
is left in it while inappropriate cleaning carried out. Tools and equipments shall be
used only for production process. Producer shall use different tools and
equipments for household purposes (Thailand Ministry of Public Health, 2012;
Winarno & Surono, 2004).
3a.3. Control of Production Process
As much as 20% of Chaiyo Farm’s production process control was in line
with Thai Primary GMP. Vessels for packaging and transportation of ingredients
in food production were suitable. A cardboard was used for packaging vessel,
whereas a large plastic bowl was used for transportation ingredients.
Non-conformity of Chaiyo Farm’s production process control was 80% of
4 points assessed aspects. Washing raw mushroom step was removed from
production process. Food processor did not wash her hands before starting
production process. Packaging material was stored at inappropriate and less
sanitation place. Production area was not sanitized before starting preparation,
production, and packaging process. Cooling process was exposed with
environment near the road. Product was stored in refrigerator with other foods and
raw materials. Processor took the final product for sensory testing by her hands

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(without clean spoon). Packaging that used did not have sealed. Water used in the
processing was not cleaned.
Washing or cleaning step is necessary to remove any contaminants of raw
materials, such as soil (US FDA, 1996). Hand washing before handling
production process prevent cross contamination from food handler to raw
materials or final product. Less sanitation and inappropriate place for storing
packaging cannot assure hygiene of packaging. Insect, rodent, and other pest can
also contaminate packaging in storing place. High amount of dust will bring any
contaminants to final product. Storing final product with raw materials and taking
food sample by hand will cause the cross contamination of final product.
Packaging without sealed can be open during transportation. It will enhance the
risk of contamination to Nam Prik Mushroom during transportation to storing
place or selling place.
There are several ways to minimize water absorption and reduce initial
microbial load of mushroom, without disappearing washing step. Food handler
can add soaking or blanching steps in the production process after washing step.
Soaking step can be carried out with adding citric acid solution.
Citric acid is commonly used as acidulant in food and beverage industry. It
is used not only as acidulant, but also as preservative (Hui & Khachatourians,
1995). Treatment with solution of 40 g/l citric acid for 10 minutes can be used to
reduce initial microbial load. Adding citric acid in soaking water does not
influence the taste of mushroom product.
Blanching process can decrease the amount of microbial, prevent
enzymatic browning, induces contraction in size and air leaking. Blanching time
of 150 seconds gave minimum volumetric shrinkage and weight loss. Enzymatic
activity was not detected on this time. Fresh mushroom that has obtained this
process can also be stored in the freeze temperature. (Vullioud et.al., 2011).
Producer should make an SSOP and SOP for her employee to make sure
good sanitation and right procedures. A special place to store packaging material
must be provided. Production and storage area shall be clean and sanitized every
day. Cooling process should be carried out in enclosed area. Producer should
provide a special refrigerator for storing final product. Food handler shall use a
clean spoon to test the organoleptic of final product. Packaging should be changed
to other type of packaging that has a sealed (Thailand Ministry of Public Health,
2012; Winarno & Surono, 2004).
3a.4. Sanitation
Chaiyo Farm’s sanitation did not comply with Thai Primary GMP as much
as 100%. Water used in the factory did not obtain any treatments or cleaning
process. There was a trash can, but trash elimination system was not proper. Trash
would be thrown per 3 days or more, depending on the amount of trash in can.
Sanitation of toilet was inappropriate. Sink for hand washing was not available.
There was no schedule for cleaning and sanitizing toilet. Waste water flowed to
public sewage without going through internal pipeline.
Water that has obtained the treatment and cleaning will ensure water is
free from pathogen bacteria. Trash stored several days in trash can, inappropriate
of sanitation toilet, can be the good sources of growing microorganisms and
produce a bad smell. Schedule for cleaning and sanitizing toilet is necessary to

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ensure sanitation toilet. Waste water flowed without internal pipeline will litter the
yard surface.
Producer shall be clean and treated according to government standards.
Trash shall be thrown to laystall at least once per day. A cleaning and sanitizing
schedule for toilet is needed. Producer shall provide a fully equipped hand
washing sink in the production area, such as flowing water, hand dryer/ tissue,
liquid soap, disinfectant dilution, and trash can with cover. Internal pipeline shall
be built (Thailand Ministry of Public Health, 2012; Winarno & Surono, 2004).
3a.5. Cleaning and Maintenance
Cleaning and maintenance of Chaiyo Farm was not complied with Thai
Primary GMP as much as 100% of 3 assessed aspects. The cleaning program was
only done once per day after production process. There was no assessment and
maintenance of tools and equipments. Producer stored chemical agent for cleaning
production area in near the sink for cleaning raw materials, inside production area.
Cleaning before starting production reduces dust or soil in the production
building. Dust and soil are the source of microbial and its spore contamination.
Assessment and maintenance of tools and equipments aims to maintain they work
in good performance at all times. Chemical agent stored in production area has a
risk to contaminate raw materials or food product.
The processor shall be clean production building before and after
production process. The producer should provide the special equipment for
cleaning production area that not used on other areas. The producer shall make a
schedule for food handler to inspect and maintenance the tools and equipments. It
shall be provide a special place for storing chemical agents that isolated from
production area (Thailand Ministry of Public Health, 2012; Winarno & Surono,
2004).
3a.6. Personnel and employee hygiene
Chaiyo Farm’s personnel and employee hygiene was complied with Thai
Primary MGP as much as 25% of 8 assessed aspects. Worker and personnel that
enter production area were not infected any communicable or contagious disease.
The owner always asked to the worker about her health. If she was sick or had
contagious disease, she was not permitted to handle all the production process.
She had to take a rest until she felt better. During that time, the owner took over
the responsibility of food handler. Food handler did not wear jewelry while
working and keep their hands and nails healthy and clean at all times.
As much as 75% of 8 personnel and employee hygiene assessed aspects
were not conformed of the regulation. Food handler did not change her clothe
with clean and proper clothe when she would enter production area. Food handler
did not worn shoes. Food handler did not wash her hand after handling other
things. There was no a SSOP to wash hands. Food handler did not use head
covering at all times in the production area. Personnel smoking in the production
area was still found. Visitor or other personnel could enter to production area
without a specific rule.
No proper clothe can disturb food handler movement. Contamination to
product may happen while food handler wear no clean clothe during production
process. SSOP is used as a guidance for employees to wash their hands in right

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way. Good hand washing procedure will decrease the risk of contamination from
employee. Head covering has function to prevent hair falled into food product.
Personnel or visitor with inappropriate behavior and without the specific rule
when entering production area, increase the risk of contamination either physical
or chemical.
Producer shall provide clean and proper clothe for food handler every day.
A SSOP for changing clothe before starting production process is necessary.
Producer shall provide shoes to be worn in the production facility. An SSOP for
washing hand before and after work is necessary. Head covering shall provide and
use while food handler is in the production area. Producer shall warn all worker
and personnel who will enter production area for not expressing inappropriate
behavior. The producer shall implement a rule (SOP) for all workers/ personnel/
visitors that want to enter the production area (Thailand Ministry of Public Health,
2012; Winarno & Surono, 2004).

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4a. CONCLUSION & RECOMMENDATION
4a.1. Conclusion
Assessment of hygienic and manufacturing practices at Chaiyo Farm
resulted in total conformity of 25.83% based on Thailand Primary Good
Manufacturing Practices. Tools, machinery, and production equipment in the
factory had complied with the regulation as much as 100%.
4a.2. Recommendation
Producer shall improve location and production buildings, control of
production process, sanitation, cleaning and maintenance, personnel and employee
hygiene to implement GMP in accordance with the Thailand Primary GMP.
Washing with soaking in the citric acid or blanching step of raw mushroom is
necessary to reduce physical contaminant and initial microbial load. Packaging
should be changed to seal packaging. After GMP implemented properly, producer
should consider implementing HACCP in the processing. Researcher has analyzed
hazard and determined critical control point (CCP) of Chaiyo Farm factory
(ICMSF, 2005; Winarno, 2012). See appendix 10 and 11 to find out hazard
analysis and CCP.

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2b. MATERIALS AND METHODS
This study was conducted in December 2nd until December 13th 2013. The
study location was Chaiyo Farm, Suratthani, Thailand. All the samples that
collected from Chaiyo Farm were analyzed in the laboratory of Food Technology,
Faculty of Science and Industrial Technology, Prince of Songkla University,
Suratthani and Hat Yai Campus, Thailand. Methodology used in the study consist
of assessment, product preparation, sample collection, physicochemical analysis,
microbiological analysis, and statistical analysis (Simon et.al., 2005).
Physicochemical and microbiological were carried out per 3 storage days at room
temperature (29oC).
2b.1. Product Preservation Using Propyl-paraben and Calcium Propionate
Three plastics of Schizophyllum commune (each plastic contains 1 kg
mushroom) were taken out from refrigerator in mushroom farm office when
production process would be started. Each mushroom was sliced to be a small
piece. If rotten part of mushroom was founded, it was separated. Sorted
mushroom was roasted for 10 minutes directly without washing process. When
the time was attained, roasted mushroom was put in clean large bowl.
Afterwards, food handler prepared curry paste. Ingredients used in this
process were salt, sugar, dried shrimp, garlic, onion, black pepper, green chili,
dried red chili, lemon grass, turmeric, galingale, galangal, fish sauce, monosodium
glutamate (MSG), and soy oil. All ingredients were crushed using a blender until
smooth texture obtained.
Curry paste was fried with soybean oil until its color change and releasing
delicious smell. Roasted mushroom was added in that frying pan. Stir-frying
process was carried out for 30 minutes. A half of frying mushroom (1500 gram)
was picked and cooled for 20 minutes in large plate. The rest of mushroom in the
frying pan was added food preservatives, 750 ppm of propyl-paraben and 1500
ppm of calcium propionate. Prior to adding food preservatives into mushroom, it
was diluted in 20 ml water. Frying was continued for 10 minutes. Fried mushroom
with preservatives was picked and cooled for 20 minutes.
Cooled curry mushroom was packed into a small packaging that made
from polyetilen (PE). It consisted of a cup and a cover. After packaging process
was done, it already became curry Nam Prik Mushroom product. See appendix 1
to know curry Nam Prik Mushroom making process.
2b.2. Analysis
2b.2.1. Sample Collection
Researcher collected twelve cups of Nam Prik Mushroom randomly from
two lots food production, non-preserved and preserved mushroom. Six cups had
30 g of sample (for microbiological analysis) and other six cups had 100 g of
sample (for physicochemical analysis). It was stored in room temperature (29oC)
inside a laboratory cupboard. Every analysis per 3 storage days took one cup of
sample. First sample was used at 12 hours after arrival in the laboratory.

14

2b.2.2. Physicochemical Analysis
2b.2.2.1. Measurement of pH value
This value was measured by pH-meter Docu-pH+ meter®, model Sartorius.
Before used, the calibration was carried out by buffered-water with pH value 4, 7,
and 10. Each sample was weighed 1 gram and put into test tube. As much as 10
ml distilled water (1/10 w/w) was added, then mixed by spoon (Hocaoglu et.al.,
2012). The measurement was carried out in 3 replicates.
2b.2.2.2. Water Activity
Every sample was measured for 5 times. The analysis was carried out by
AquaLab® Model Series 3, Serial No. 01107060B. One time measurement used
approximately 5 grams of sample. It put into a small plastic container and was
placed to the instrument chamber. The measurement was conducted in five
replicates (Aishah & Rosli, 2013).
2b.2.2.3. Color
Approximately 25 grams of sample at random was put into quartz pot. It
was measured using MiniScan XE PLUS ® Model No.45/0-L, Serial No. 7097.
This instrument defined colour numerically in terms of its lightness or “L” value
(black = 0, white = 100), “a” value (greenness 0 to -100, redness 0 to +100) and
“b” value (blueness 0 to -100, yellowness 0 to +100). Illuminant D65 and 10
Observer were used in the active view option. To measure the colour of product,
took several spoon of it, placed on a glass cup, then click “Start” on the desktop of
computer. The measurement was carried out as much as ten replicates for each
sample (Kim et.al., 2005).
2b.2.3. Microbiological Analysis
2b.2.3.1. Media & Chemical
These analysis need several media and chemical:
a. Butterfield’s Phospate-Buffered Dilution Water
Stock Phosphate Buffer Solution: As much as 34 g potassium dihydrogen
phosphate, KH2PO4, was dissolved in 500 ml distilled water. The value of pH
solution should be adjusted to 7.2 with 1 N NaOH, and diluted to 1 liter. As
much as 1.25 ml stock phosphate buffer solution was added. Volume of this
solution brings to 1 liter with distilled water. Solution was dispensed in bottle
that will provide 99 ml and/or 9 ml. Then, it was sterilized by autoclave at
121oC for 15 minutes (FDA, 2001).
b. Malt Extract Agar (MEA) Medium
Media that used in this study was ready-to-use media, Difco MEA.
c. Plate Count Agar (PCA) Medium
In making PCA, the ingredients are 5 g tryptone, 2.5 g yeast extract, 1.0 g
glucose, 15.0 g agar. All the ingredients are dissolved into 1 liter distilled
water. The value of pH solution should be adjusted at 7.0 by 1N NaOH. The
media was sterilized by autoclave 121oC, for 15 minutes (Askar & Treptow,
1993).

15

d. Ringer Rinse Solution
To prepare Ringer solution, as much as 2.15 g sodium chloride, 0.075 g
potassium chloride, 0.12 g calcium chloride, 0.5 g sodium thiosulfate were
dissolved in distilled water up to 1 liter. The solution was sterilized in
autoclave for 15 minutes, at 121oC (Askar & Treptow, 1993).
e. Alcohol 70% for sterilization hand of researcher.
2b.2.3.2. Preparation sample of final product
The microbial tests were carried out are total aerobic bacteria (Plate Count
Agar) and total yeasts & molds (Malt Extract Agar). A cup of sample (30 grams)
was taken and mixed it with sterile spoons or other utensils before withdrawing
the analytical unit from a sample. As much as 25 grams sample was weighed
aseptically. It put into sealed plastic bag. Dilution water (buffered-phospate water)
225 ml was added into plastic bag, then it stomached by hand. This became the
1:10 dilution. As much as 1 ml of the blended 1:10 dilution was added into a 9 ml
dilution blank to make the 1:100 dilutions. This procedure was repeated to prepare
serial dilutions of 1:1000. Then, it was shaken 25 times before used (Andrews
et.al., 2003).
2b.2.3.3. Total Aerobic Plate Count ( FDA BAM Method: Aerobic Plate
Count)
Dilutions were shaken. Each dilution was pipetted 1 ml into marked petri
dishes. It was made duplicates of each dilution. Plate Count Agar 12-15 ml was
poured to each plate. Sample dilutions and agar media was mixed uniformly. Let
agar solidify. Solidified petri dishes was inverted and incubated promptly for 48 ±
2 h at 35°. Counts plates containing 25-250 colonies.

where:
N
= Number of colonies per ml or g of product
∑ C = Sum of all colonies on all plates counted
n1 = Number of plates in first dilution counted
n2 = Number of plates in second dilution counted
d
= Dilution from which the first counts were obtained, (Maturin et.al., 2001).
2b.2.3.4. Total Yeasts and Molds (FDA BAM Method: Yeasts, Molds and
Mycotoxins Method)
Spread-plate method. Dilutions were shaken. Aseptically, as much
as 0.1 ml of each dilution was pipetted on pre-poured solidified MEA agar plates.
Inoculum was spread with a sterile bent glass rod. Each dilution was plated in
duplicate. Plates were incubated in the dark (inside a cupboard on PSU Suratthani
Laboratory) at 25°C for 5 days. Count plates containing 10-150 colonies.

where:
N
∑C

= Number of colonies per ml or g of product (cfu/g)
= Sum of all colonies on all plates counted

16

n1
n2
d

= Number of plates in first dilution counted
= Number of plates in second dilution counted
= Dilution from which the first counts were obtained, (Tournas
et.al., 2001).

2b.2.3.5. Culture Settling Plate Technique
To measure the microbiological air quality of Chaiyo Farm processing
area and laminar flow, media PCA and MEA were exposed on the air, in
the processing area and laminar flow for 15 minutes. Then it was
incubated as same as final product procedures. To calculate the number of
microbial (total aerobic bacteria or total yeast & mold), formula below was
used:
N = Caverage x 1
x 60 min x 168 hours x
1
15 min
1 hour
1 week
plate wide (cm2)
where:
N
= Number of colonies per ml or g of product (cfu/week/cm2)
Caverage = Average of all colonies on two plates, (Salustiano et.al., 2003;
Obadina et.al., 2009).
2b.2.3.6. Rinsing Test
It used rinse method to determine microbiological quality of
packaging. As much as 20 ml Ringer rinse solution was added to a
packaging cup and recap it aseptically. The cup was shaken for 10
minutes. It was taken 1 ml of the rinse solution and put into a plate. It was
carried out in two replicates. Desired medium (PCA and MEA) was
poured into the plate as much as 15 ml. PCA plates were incubated
promptly for 48 ± 2 h at 35°, whereas MEA plates were incubated in the
dark at 25°C for 5 days.
To calculate the number of microbial (total aerobic bacteria or total
yeast & mold), formula below was used:
N
= Caverage x rinse solution volume
where:
N
= Number of colonies per cup (cfu/cup)
Caverage
= Average of all colonies on two plates, (Askar & Treptow,
1993).
2b.2.3.7. Personal observation
Personal observation was carried out by the researcher. As much as 5
grams of sample was placed on petri dish. The researcher checked the
presence of slimy texture, off-odor, and mold appearance on the final
product (Brennan et.al., 1998).

17

3b. RESULT AND DISCUSSION
Product Evaluation
3b.1. Physicochemical (See appendix 4)
3b.1.1. Color
There was no change significantly in color parameters (L*, a*, b*) of both
non-preserved and preserved curry mushrom color during storage. A mean of
color parameter L* value (lightness) of NP and P were 37.28 and 37.07. The mean
of color parameter a* value (redness) NP and P were 9.50 and 9.66.
These results are in agreement with Batt and Tortello (2014). Addition of
preservative will not affect the color of food product due to the nature of propylparaben and calcium propionate are colorless in solution.
3b.1.2. pH value
Non-preserved and preserved mushroom had no different pH value
significantly on 0 storage day. While the product stored in packaging, proteolysis
process might had happened. In food product that contains high protein,
increasing of free amino acids content and ammonia in line with proteolysis.
Proteolysis occurs may be due to action of Pseudomonas spp (Jaye et.al., 1962; in
Mexis et.al., 2012). As a result of protein metabolism, pH value increased day
after day (Fung & Crozier-Dodson, 2008). Their finding was similar to proteolysis
of curry Nam Prik Mushroom. The increasing of pH value can be observed from
the initial pH of non-preserved curry mushroom product, 5.98 to 6.7 after 3 days
storage and 6.7 to 7.55 after more 3 days storage. For preserved curry mushroom,
the increasing pH value after 6 days storage was slightly than 3 days storage. It
was possibly caused by human error when prepared samples or crosscontamination during packaging process.
3b.1.3. Water activity
Adding preservatives did not affect the changes of water activity
significantly on 0 storage day and during storage. Non-preserved mushroom had a
mean of water activity as much as 0.948, whereas preserved mushroom had 0.938.
It is in agreement with Davidson, 2002. He said preservatives do not significantly
affect water activity.

3b.2. Microbiological (See appendix 5)
The microbiological analysis was carried out at 12 hours after the sample
was taken from the seller. The results for microbiological analysis during storage
from 2nd December 2013 until 13th December 2013 were showed in Appendix 5.
3b.2.1. Total Aerobic Count and Total Yeast & Mold
All results of total aerobic plate count were spreaders (See appendix 6).
These spreaders were contamination by yeast colonies. Possibly, preservatives
were more reduced a number of bacteria and mold than yeast at product’s pH 6.03

18

(Obadina et.al., 2010). A number of yeasts and molds increased significantly on 3
days storage for both of samples (See appendix 7).
Calcium propionate is more effective to inhibit molds, little effective to
inhibit bacteria, and no activity to against yeasts at pH 4.5, 6.0 or slightly higher
for some foods (Furia, 1972). Besides, propyl paraben is more effective to against
bacteria at pH 6.8 to 7.0 (Davidson et.al., 2005).
Microbiological quality of packaging product and the air of laminar flow
had total aerobic plate count less than 1 cfu/cup and 1 cfu/week/cm2. The air in
production plant had total aerobic plate count as much as 11 x 10 2 cfu/week/cm2
(see appendix 8). Total yeast and mold of the production plant air was spreader.
Microbiological quality of production plant air, either total aerobic bacteria or
total yeast & mold, was higher than 3 x 101 cfu/week/cm2- American Public
Health Association (APHA) standards (Salustioano et.al., 2003).
From microbiological quality of packaging, the air of laminar flow and
production plant; recontamination of final product could happen after cooking
process. It was liked cooling final product in a room which has inappropriate air
condition and inappropriate wrapping technique of final product. To prevent
recontamination, producer should implement good handling and sanitary
conditions. It also can reduce high initial microbial load of raw materials (Bates
et.al., 2001). Fully covered production plant is necessary as well.
pH is very important factor affecting chemical preservatives activity such
as weak organic acid. Weak organic acid activity is influenced by undissociated
molecule or protonated form. Reduction pH of food can increase in the propotion
of undissoated molecule (Davidson, 2002; Rico-Munoz & Davidson, 1983 in
Davidson, 2002). Curry mushroom products, either non-preserved or preserved,
had pH value on range 5.80-6.03 on 0 day storage. According to Marin
et.al.(2002), 0,3 % calcium propionate did not perform inhibiting mold growth
rate at pH 6.0. However at pH 4.5, calcium propionate activity was most effective
to inhibit xerophilic molds at any aW level.
Propyl paraben defend inhibiting activity at a wide range of pH values. Up
to pH 8.5, it maintains molecule to retain undissoated. One of factors affecting its
activity is product composition. Food containing high fat will decrease propyl
paraben activity due to it soluble in lipid phase. On the other word, it reduces the
availability of propyl paraben to against microorganisms in water phase
(Davidson, 2002; Davidson et.al., 2005; Lou &Yousef, 1999). It possibly
decreased propyl paraben activity in curry paste mushroom product. Curry paste
mushroom was added 5 tablespoons soybean oil while it was made.
3b.2.2. Personal Observation (See appendix 9)
Slimy texture, off-odor, and mold appearance were found since 3 storage
days either on preserved or non-preserved Nam Prik Mushroom. It increased on 6
storage days. The slime was seen on the surface of curry Nam Prik Mushroom. It
was formed due to the combination of effect from proteolysis and amylolytic
enzymes that produced by spoilage bacteria. Off-odor that produced of curry
Nam Prik Mushroom was like ammonia. It was a result of protein breakdown
(Saranraj & Geetha, 2012). Spoilage mold was appeared on the curry Nam Prik
Mushroom surface. It was indicated as a white spot.

19

Preservatives cannot optimally inhibited microorganisms on the product
that contained high microbial load. Therefore, either product aroma of preserved
or non-preserved curry mushroom decreased slightly and gave off-odor. Preserved
and non-preserved curry mushroom cannot be accepted since 3 days storage.
It is different with Lohano et.al., 2010. Calcium propionate (0.4 grams)
added to bread and carried out a sensory evaluation (color, texture, and aroma).
The bread that contained calcium propionate and stored in room temperature had
longer shelf life 1 day than bread without this preservative (as control).

20

21

4b. CONCLUSION & RECOMMENDATION
4b.1. Conclusion
Propyl-paraben and calcium propionate did not influence pH, color, and
water activity of Nam Prik Mushroom. The effectiveness combination of propylparaben and calcium propionate to extend the shelf life of curry mushroom was
not proven. Either non-preserved or preserved Nam Prik Mushroom could not be
accepted organically after 3 days storage at room temperature (29 oC).
4b.2. Recommendation
Used of combination of propyl-paraben and calcium propionate can be
changed to the use of other food preservatives. It should be able to work in food
with high pH and high fat content as well. It should be very active inhibit the
mold. Ready-to-eat Nam Prik Mushroom should be changed to be ready-to-serve
food product. Before serving Nam Prik Mushroom, consumer shall reheat this
product. In the initial step, surface of cooking pan is coated by cooking spray or
vegetable oil. Then, Nam Prik Mushroom is reheated with stirring about 3
minutes on the top of stove that has over medium-high heat (Wholefoods Market,
2013).

22

23

REFERENCES
Aishah, M.S., W.I. Wan Rosli. 2013. Effect of different drying techniques on the
nutritional values of oyster mushroom (Pleurotus sajor-caju). Sains
Malaysiana 42(7)(2013):937-941.
Andrews, Wallace H., Thomas S. Hammack. 2003. Bacteriological analytical
manual chapter 1: food sampling and preparation of sample homogenate
[internet].
[referred
on
August
16th
2013].
Available
in:.
www.fda.gov/Food/FoodScienceResearch/LaboratoryMethods/ucm063335.ht
m.
Askar, Ahmed, Hans Treptow. 1993. Quality Assurance in Tropical Fruit
Processing. Berlin: Springer-Verlag Berlin Heiderberg.
Bates, R.P., J.R. Morris, P.G. Crandall. 2001. Principles and Practices of Smalland-Medium Scale Fruit Juice Processing. Rome: FAO.
Batt, Carl A., Mary Lou Tortorello. 2014. Encyclopedia of Food Microbiology.
London: Academic Press. ISBN: 978-0-12-384730-0.
Brennan, Martine H., T. Ronan Gormley. 1998. Extending the shelf life of fresh
sliced mushrooms. Ballsbridge: Teagasc.
[CFIS] Canadian Food Assessment System. 2013. Food Retail and Foodservices
Code. Canada: Alberta Health and Wellness.
Davidson, P. Michael. 2002. Control of Foodborne Microorganisms. Edited by
Vijay K. Juneja & John N. Sofos. New York: Marcel Dekker, Inc.
Davidson, P. Michael, , John N. Sofos, A.L. Branen. 2005. Antimicrobials in
Food, Third Edition. Edited by P. Michael Davidson, John N. Sofos, A.L.
Branen. Boca Raton: CRC Press, Taylor & Francis Group.
Dias, Maria Angelica Costa, Anderson S. Sant’Ana, Adriano G. Cruz, Jose de
Assis F. Faria, Carlos Augusto Fernandes de Oliveira, Evandro Bona. 2012.
On the implementation of good manufacturing practices in a small processing
unity of mozzarella cheese in Brazil. Food Control 24 (2012) 199-205. doi:
10.1016/j.foodcont.2011.09.028.
[FDA] Food and Drug Administration. 2001. Bacteriological analytical manual:
buffered-phosphatewater [internet]. [referred on August 15th 2013]. Available
in : www.fda.gov/Food/FoodScienceResearch/LaboratoryMethods/ucm061208.htm.
Fung, Daniel Y. C., Beth Ann Crozier-Dodson. 2008. Tempeh, a mold modified
indigenous fermented food. in Handbook of Fermented Functional Foods,
Second Edition. editor: Edward R. Farnworth. Boca Raton: CRC Press.
Furia, T.E. 1972. CRC Handbook of Food Additives 2 nd Edition Volume I.
Danvers: CRC Press LLC.
Hui, Y.H., George G. Khachatourians. 1995. Food Biotechnology:
microorganisms. Canada: Wiley-VCH.
Hocaoglu, Ash, Ahmet Sukru Demirci, Tuncay Gumus, Mehmet Dermici. 2012.
Effects of gamma irradiation on chemical, microbial quality, and shelf life of
shrimp. Radiation Physics and Chemistry 81 (2012) 1923-1929.
[ICMSF] International Commision on Microbiological Specifications for Foods.
2005. Microorganisms in Foods 6 Second Edition, Microbial Ecology of Food

24

Commodities. New York: Kluwer Academic/ Plenum Publishers. eISBN: 0306-48676-8.
[Indonesia Ditjen PPHP] Indonesia Direktorat Jenderal Pengolahan dan
Pemasaran Hasil Pertanian. 2009. Guideline of Good Manufacturing Practices
of Agricultural Product. Jakarta: Direktorat Mutu dan Standarisasi,
Departemen Pertanian.
Jaye, M., Kittaka, R. S., Ordal, Z. J. 1962. The effect of temperature and
packaging material on the storage life and bacterial flora of ground beef. Food
Technology, 16, 95-98.
Kim, Ki Myong, Jung A Ko, Jin Sil Lee, Hyun Jin Park, Miford A. Hanna. 2005.
Effect of modified atmosphere packaging on the shelf-life of coated whole and
sliced mushrooms. LWT 39 (2006) 364-371.
Lohano, Dileep Kumar, Saghir Ahmed Sheikh, Muhammad Shahnawaz. 2010.
Effect of chemical preservatives on the shelf life of bread at various
temperatures. Pakistan Journal of Nutrition 9 (3): 279-283, 2010. ISSN: 16805194.
Lou, Y., A.E. Yousef. 1999. Listeria, Listeriosis, and Food Safety; Second
Edition, Revised, and Expanded. Edited by Elliot T. Ryser & Elmer H. Marth.
New York: Marcel Dekker, Inc.
Marin, S., M.E. Guynot, P. Neira, M. Bernado, V. Sanchis, A.J. Ramos. 2002.
Risk assessment of the use of sub-optimal levels of weak-acid preservatives in
the control of mould growth on bakery products. International Journal of Food
Microbiology 79 (2002) 203-211.
Maturin, Larry, James T. Peeler. 2001. Bacteriological analytical manual chapter
3 aerobic plate count [internet]. [referred on August 13th 2013]. Available
in:.www.fda.gov/Food/FoodScienceResearch/LaboratoryMethods/ucm063346
.htm.
Mexis, S.F., E. Chouliara, M.G. Kontominas. 2012. Shelf life extension of ground
chicken meat using an oxygen absorber and a citrus extract. LWT- Food
Science and Technology 49 (2012) 21-27. doi: 10.1016/j/lwt.2012.04.012.
Msagati, Titus A. M. 2012. Chemistry of Food Additives and Preservatives. John
Willey & Sons Ltd.
Obadina, A. O., O. B. Oyewole, L. O. Sanni, K. I. Tomlins, A. Westby. 2009.
Improvement of the hygienic quality of wet ‘fufu’ produced in South West
Nigeria.
Food
Control
21
(2010)
639-643.
doi:
10.1016/j.foodcont.2009.09.009.
Ray, Bibek. 2005. Fundamental Food Microbiology 3th Edition. CRC Press:
Florida, United States.
Rico-Munoz, E., P.M. Davidson. 1983. The effect of corn oil and casein on the
antimicrobial activity of phenolic antioxidants. Journal Food Science 48:12841288, 1983.
Salustiano, Valeria Costa, Nelio Jose Andrade, Sebastiao Cesar Cardoso Brandao,
Raquel Monteiro Cordeiro Azeredo, Sandra Aparecida Kitakawa Lima. 2003.
Microbiological air quality of processing areas in a dairy plant as evaluated by
the sendimentation technique and a one-stage air sampler. Brazilian Journal of
Microbiology (2003) 34:255-259.

25

Saranraj, P., M. Geetha. 2012. Microbial Spoilage of Bakery Products and Its
Control by Preservatives. International Journal of Pharmaceutical &
Biological Archives 2012; 3(1):38-48. ISSN: 0976-3333
Schimdt, Olaf. 2006. Wood and Tree Fungi; Biology, Damage, Protection, and
Use. Heidelberg: Springer.
Simon, Ana, Elena Gonzales-Fandos, Vanesa Tobar. 2005. The sensory and
microbiological quality of fresh sliced mushroom (Agaricus bisporus L.)
packaged in modified atmospheres. International Journal of Food Science and
Technology 2005, 40, 943-952. doi: 10.1111/j.1365-2621.2005.01028.x.
Smith, Jim, Lily Hong-Shum. 2011. Food Additives Data Book. Great Britain:
Blackwell Publishing Ltd. 0-632-06395-5.
Thailand Ministry of Public Health. 2012. RE: Manufacturing Procedures,
Production Equipment and Appliance, and Food Storage of Pre-packaged
Processed Foods. Thailand Ministry of Public Health, (NO 342) B.E. 2012.
Tournas, Valerie, Michael E. Stack, Philip B. Misli