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 *Corresponding author.

Email: nazmimat@upm.edu.my

eISSN: 2550-2166 / © 2018 The Authors. Published by Rynnye Lyan Resources

  Food Research 3 (2) : 177 - 181 (April 2019)
  Journal homepage: http://www.myfoodresearch.com

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Field performance comparison of the combine harvesters utilized for rice

harvesting in Malaysia

1 Wagiman, N.A., 1, 2, 3 *Nawi, N.M., 1, 3Yahya, A, 2, 4Su, A.S.M. and 1Nasir, R.M.

1Department of Biological and Agricultural Engineering, Faculty of Engineering, Universiti Putra

Malaysia, 43400, UPM Serdang, Selangor, Malaysia
2Smart Farming Technology Research Centre, Faculty of Engineering, Universiti Putra Malaysia, 43400,

UPM Serdang, Selangor, Malaysia
3Institute of Plantation Studies, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia

4Department of Agriculture Technology, Faculty of Agriculture, Universiti Putra Malaysia, 43400 Serdang,

Selangor, Malaysia

Article history:

Received: 9 July 2018

Received in revised form: 14

November 2018

Accepted: 13 November 2018

Available Online: 20

November 2018

Keywords:

Field performance,

Large combine harvester,

Mini combine harvester,

Field capacity,

Rice production

DOI:
https://doi.org/10.26656/fr.2017.3(2).136

Abstract

A large combine harvester has long been used for harvesting rice in Malaysia. Recently, a

mini combine harvester has been introduced into this industry to be a low-cost alternative

for a large combine harvester. Thus, this study was carried out to compare the field

efficiency (FE), effective field capacity (EFC), fuel consumption (FC), and field machine

index (FMI) between a mini combine harvester (MCH) and large combine harvester

(LCH) under similar field conditions. The field performances of the combine harvesters

were measured during harvest operation in two consecutive seasons. The EFC for both

MCH and LCH in harvest operation were found to be 0.91 and 1.30 ha/hr, respectively. In

terms of the FE, MCH had 0.78% higher efficiency than LCH. For the FMI, LCH and

MCH had 0.86 and 0.87, respectively. For the FC, LCH consumed more fuel (14.51 L/ha)

as compared to MCH (14.25 L/ha). T-test statistical analysis showed that there was no

significant difference between LCH and MCH for FE, EFC, FMI and FC. The results

suggested that MCH was more efficient and economical in conducting the harvest

operation in a rice field.

1. Introduction

Rice is one of the most important crops in Malaysia

because it is a staple food for the people in the country

(Ramli et al., 2012). Malaysian farmers supply 1.69

million metric ton of rice which were harvested from

675,000 hectares of rice granaries throughout the

country, with an average yield of 2.50 metric ton per

hectare in 2011 (Wailes and Chaves, 2012). However,

due to the total domestic consumption of rice is

estimated to be around 2.3 million ton yearly, the current

rice self-sufficiency level (SSL) in Malaysia is only

fulfilling 73% of the target (Raziah et al., 2010). Due to

that, this country still needs to import rice from other

countries, such as Thailand, Vietnam and India.

Harvesting is one of the most important operations in

rice production. Traditionally, this operation was

regarded as a labor-intensive operation in the process of

rice cultivation (Bautista, 2005). However, rice

production in Malaysia faces the labor shortages problem

due to the difficulty of getting people who are willing to

work in this sector (Adam and Pebrian, 2017). Therefore,

a combine harvester has long been introduced to

mechanize rice harvesting system in an effort to tackle

the labor shortage in Malaysian rice production. A well-

designed combine harvester can play an important role in

harvesting rice in time, efficiently and at low cost.

Typically, rice harvesting contractors use a large

combine harvester to harvest rice in Malaysia. However,

the use of a large and heavy combine harvester in a rice

field could damage the soil and break the hardpan.

Recently, several rice harvesting contractors have

initiated the use of a mini combine harvester which is

smaller and lighter than the large combine harvester.

However, there is no scientific study was undertaken to

compare the performance of these combine harvesters in

Malaysia. Bawatharani et al. (2015) claimed that the

performance of any combine harvester varied with

respect to the field and the machine operational

conditions. Thus, several investigations with different

types of paddy varieties at various field conditions were

needed to study the performance of different types of

178 Wagiman et al. / Food Research 3 (2) (2019) 177 - 181

eISSN: 2550-2166 © 2018 The Authors. Published by Rynnye Lyan Resources

combine harvesters to optimize the operational factors

(Chegini, 2013; Hossain et al., 2015).

Therefore, the purpose of this study was to compare

the field performances between a mini and large combine

harvesters under similar field conditions in Malaysia.

The specific objective of this study was to measure the

field efficiency (FE), effective field capacity (EFC), fuel

consumption, and field machine index (FMI) of both

types of the combine harvesters.

2. Materials and methods

2.1 Study area

This research was conducted at two different rice

fields (Field A and Field B), located at Jitra, Kedah,

Malaysia. Each field was divided into two equal plots

namely plot 1 and plot 2. One plot was allocated for a

mini combine harvester (MCH) and another plot for a

large combine harvester (LCH). After identifying the

location of the site, the coordinate of each location was

taken using GPS Trimble Juno 3b. The experiments were

conducted in two consecutive harvest seasons.

2.2 Combine harvester

There were two types of combine harvesters used in

this study namely LCH and MCH. There were two

combine models were chosen for the LCH namely New

Holland 1545 (Figure 1) and New Holland 8070 (Figure

2). For the MCH, it was represented by Kubota DC-95M

(Figure 3). The selection of these three different models

was made since these models were predominantly used

by contractors in this state. Table 1 shows the

information of study areas with their respective combine

harvesters. The specifications of combine harvesters are

shown in Table 2.

2.3 Methods

2.3.1 Measurement of the operation times

Time consumption during harvest operation was

manually measured using a stopwatch. The times that

were recorded including the starting and stopping time.

Duration for the combine harvester to perform straight

driving and cornering were also recorded. This data was

important to calculate the time losses during harvest

operation (Sattar, 2015).

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Table 2. Specification of the combine harvesters

Specification
Type of Combine Harvester

New Holland 1545 New Holland 8070 Kubota DC-95M

Maximum engine power, kW 96 103 75

Implement width, m 3.58 4.57 2.48

Total body mass, kg 6450 8240 3550

Footprint area, m2 1.52 1.07 1.95

Total contact pressure, kg/m2 4243.42 7700.93 1820.51

Total contact area pressure, kPa 41.61 75.52 17.85

Location Plot Model of Combine Area (ha)

Field A
1 New Holland 1545 0.39

2 Kubota DC-95M 0.39

1 New Holland 8070 0.74
Field B

2 Kubota DC-95M 0.74

Table 1. Machinery information for the study area

Figure 1. Typical large New Holland combine harvester

model 1545

Figure 2. Typical large New Holland combine harvester model

8070

Figure 3. Typical mini Kubota combine harvester model DC-

95M

  Wagiman et al. / Food Research 3 (2) (2019) 177 - 181 179

eISSN: 2550-2166 © 2018 The Authors. Published by Rynnye Lyan Resources

2.3.2 Determination of fuel consumption

The fuel of the combine harvesters was fully refilled

before harvesting operation was started. The amount of

fuel consumption of the combine harvesters was

determined by measuring the difference of the fuel inside

the fuel tank before and after the operation (Abdallah,

2008). In order to have comparable data on fuel

consumption, a gear combination of each combine

harvester was also recorded.

2.3.3 Determination of field efficiency

Field efficiency (FE) was defined as the percentage

of time when the machine is operated at its full rated

speed and width in the field (Nasri, 2015). FE described

how effective the time was spent to do the work (Grisso,

2014). Because of the headland turns, machine trouble,

ground surface and overlapping, the FE for an actual

field operation was always less than 100% (Zandonadi,

2012). FE was determined as follows:

Where FE = field efficiency (%); EFC = effective field

capacity (ha/hr); TFC = theoretical filed capacity (ha/hr)

The ability of a combine harvester to harvest rice

under an actual field condition can be defined as an

effective field capacity (EFC) (Zhou, 2012). EFC was

calculated using the formula:

Where A = area covered (ha); Tp = productive time (hr);

and T = non-productive time (hr) such as breakdown in a

field

2.3.4 Determination of field machine index

Field machine index (FMI) was the index that

indicates the turning effectiveness of a combine

harvester. As the value of FMI was higher, it was

indicated that the turning time of the combine harvester

was low (Shamshiri et al., 2013)

Where EOT = effective operation time in second.

3. Results and discussion

3.1 Field efficiency

The averages of FE from two measurements in two

seasons were shown in Figure 4. It was found that the FE

for the MCH and LCH were 86.53% and 85.75%,

respectively. The FE for the MCH was 0.78% higher

than the LCH, indicating that the MCH more efficient in

terms of time consumption in conducting harvesting

operation. This finding is supported by the fact that the

FE increases as the width of the implement decrease

(Hanna, 2016). In this study, the widths of the cutter bar

for the MCH and LCH were about 2.48 m and 4.57 m,

respectively. According to t-test results shown in Table

3, the value of t stat and t critical two-tail were -0.21 and

3.18, respectively. There was no significant difference

between LCH and MCH in terms of FE since the value

of t stat was lower that the value of t critical two tail.

3.2 Effective field capacity

The averages of EFC from field measurements in

two seasons were shown in Figure 5. Figure 5 shows that

the EFC of the LCH was 1.30 ha/hr, 0.39 ha/hr (30%)

higher than the EFC for the MCH of 0.91 ha/hr. This is

acceptable because the MCH has a shorter cutter bar as

compared to the LCH. Hossain et al., (2015) reported

that the EFC of a combine harvester would decrease

when the width of the implement decreased. According

to t-test results shown in Table 4, the value of t stat and t

critical two-tail were 2.84 and 3.18, respectively with

0.05 alpha value. This indicates that there was no

significant difference between LCH and MCH in terms

of EFC since the value of t stat was lower than the value

of t critical two tail.

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(1)

(2)

(3)

Figure 4. Field efficiency for harvesting operation

Field Efficiency (FE)

Type of combine LCH MCH

Mean 85.75 86.53

Variance 4.92 66.45

T-Stat -0.21

P(T<=t) two-tail 0.85

T Critical two-tail 3.18

Figure 5. Effective field capacity for harvesting operation

Table 3. T-test results for field efficiency

180 Wagiman et al. / Food Research 3 (2) (2019) 177 - 181

eISSN: 2550-2166 © 2018 The Authors. Published by Rynnye Lyan Resources

3.3 Fuel consumption

Figure 6 shows that LCH consumed 14.51 l/ha of

fuel while MCH only consumed 14.25 l/ha of fuel. This

data indicated that the LCH consumed 0.26 L/ha fuel

more than MCH under typical field conditions. This is

true because a bigger engine will typically consume

more fuel. In this study, based on Table 2, is it proven

that the engine power of the LCH was higher than the

MCH thus it consumed more fuel. According to t-test

results shown in Table 5, the value of t stat and t critical

two-tail were 0.13 and 3.18, respectively with the alpha

value of 0.05. There was no significant difference

between LCH and MCH for fuel consumption since the

value of t stat was lower that the value of t critical two

tail.

3.4 Field machine index

According to the data shown in Figure 7, field

machine index (FMI) for the MCH was 0.87 while for

the LCH was 0.86. The MCH was found to have

relatively higher FMI value as compared to the LCH.

This is because the MCH needed to do more cornering as

compared to the LCH due to its shorter cutter bar.

However, it was observed that the MCH could have

higher field speed as compared to LCH. According to t-

test results shown in Table 6, the value of t stat and t

critical two-tail were -0.21 and 3.18, respectively with

alpha value of 0.05. There was no significant difference

between LCH and MCH for FMI since the value of t stat

was lower the value of t critical two tail.

4. Conclusion

The EFC for the both LCH and MCH in harvest

operation were 1.30 ha/hr and 0.91 ha/hr, respectively. In

terms of FE, the MCH had 0.78% higher efficiency as

compared to the LCH. Meanwhile, the FMI for the LCH

and MCH were 0.86 and 0.87, respectively. For the FC,

the LCH consumed 0.26 L/ha more fuel than the MCH.

The results suggested that the MCH was more efficient

in conducting the harvest operation in a rice field.

Besides that, the MCH consumed less fuel than the LCH.

The time loss of the MCH in conducting the harvesting

was less as compared with the LCH since FMI values for

the MCH was the highest. However, statistically, there

were no significant differences between the LCH and

MCH in terms of FE, EFC, FC and FMI.

Acknowledgments

The authors acknowledge research grant from the

Ministry of Agriculture Malaysia, (MOA) through Muda

Agricultural Development Authority (MADA). The

authors also acknowledge technical supports from the

staff of the Department of Mechanical and Electrical

(BME), MADA. The authors also acknowledge financial

support from Universiti Putra Malaysia through Putra

Grant – Graduate Research Initiative Scheme (Vot no:

9606300).

References

Abdallah, B.A. (2008). Effect of Ploughing Depth and

Speed of Three difference tractors. Khartoum,

Sudan: University of Khartoum, BSc. Thesis.

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Table 6. T-test results for field machine index

Table 5. T-test results for fuel consumption

Table 4. T-test results for effective field capacity

Effective Field Capacity (EFC)
Type of combine LCH MCH

Mean 1.30 0.91

Variance 0.04 0.01

T Stat 2.84

P(T<=t) two-tail 0.07

T Critical two-tail 3.18

Fuel Consumption (FC)

Type of combine LCH MCH

Mean 14.51 14.25

Variance 4.30 25.75

T Stat 0.13

P(T<=t) two-tail 0.90

T Critical two-tail 3.18

Field Machine Index (FMI)

Type of combine LCH MCH

Mean 0.86 0.87

Variance 0.00 0.01

T Stat -0.21

P(T<=t) two-tail 0.85

T Critical two-tail 3.18

Figure 7. Field machine index for harvesting operation

Figure 6. Fuel consumption for harvesting operation

  Wagiman et al. / Food Research 3 (2) (2019) 177 - 181 181

eISSN: 2550-2166 © 2018 The Authors. Published by Rynnye Lyan Resources

Adam, N.S. and Pebrian, D.E. (2017). Factors affecting

farmers’ satisfactions with mechanized rice

harvesting in Malaysian paddy fields: A case study

of hiring custom operators. Agricultural Engineering

International, 19(2), 120–128.

Bautista, E.U. and Javier, E.F. (2005). The Evolution of

Rice Production Practices the Evolution of Rice

Production Practices. Discussion Paper Series No.

2005-14. Phillipines: Philippine Institute for

Development Studies

Bawatharani, R., Dharmasena, D.N.J.D.A.N. and

Bandara, M.H.M.A. (2015). Field Performance of a

Conventional Combine Harvester in Harvesting Bg-

300 Paddy Variety in Batticaloa, Sri Lanka.

International Journal of Engineering Research, 5013

(4), 33–35. https://doi.org/10.17950/ijer/v4s1/108

Chegini, G.R. (2013). Determine of Optimum Operating

Conditions of Combine Harvester with Stripper-

Header. World Applied Sciences Journal, 23(10),

1399–1407.

Grisso, R.D., Kocher, M.F., Adamchuk, V.I., Jasa, P.J.

and Schroeder, M.A. (2004). Field Efficiency

Determination Using Traffic Pattern Indices. Applied

Engineering in Agriculture, 20(5), 563–572. https://

doi.org/10.13031/2013.17456

Hanna, M. (2016). Estimating the Field Capacity of

Farm Machines. Retrieved from Iowa State

University website: https://

www.extension.iastate.edu/agdm/crops/html/a3-

24.html

Hossain, M.A., Hoque, M.A., Wohab, M.A., Miah,

M.A.M., and Hassan, M.S. (2015). Technical and

economic performance of combined harvester in

farmers’ field. Bangladesh Journal of Agricultural

Research, 40(6), 291–304. https://doi.org/10.3329/

bjar.v40i2.24569

Nasri, N.N., Nawi, N.M., Abdullah, N., Saripa, S. and

Mat, R. (2015). Measurement of Field Efficiency for

Different Field Operations in Sweet Corn

Production. Proceedings of the Technology and

Innovation National Conference, 1–6.

Ramli, N.N., Shamsudin, M.N., Mohamed, Z. and

Radam, A. (2012). The Impact of Fertilizer Subsidy

on Malaysia Paddy / Rice Industry Using a System

Dynamics Approach. International Journal of Social

Science and Humanity, 2(3), 213–219.

Sattar, M., Ali, M., Ali, L., Waqar, M.Q., Ali, M.A. and

Khalid, L. (2015). Grain Losses of Wheat as

Affected by Different Harvesting and Threshing

Techniques. International Journal of Research in

Agriculture and Forestry, 2(6), 20–26.

Shamshiri, R., Ehsani, R., Maja, J.M. and Roka, F.M.

(2013). Determining machine efficiency parameters

for a citrus canopy shaker using yield monitor data.

Applied Engineering in Agriculture, 29(1), 33–41.

https://doi.org/10.13031/2013.42526

Wailes, E.J., and Chavez, E.C. (2012). ASEAN and

Global Rice Situation and Outlook. ADB Sustainable

Development Working Paper Series, 22, 1–42.

Zandonadi, R.S. (2012). Computational Tools for

Improving Route Planning in Agricultural Field

Operations. Lexington, Kentucky: University of

Kentucky, PhD Thesis.

Zhou, K., Leck Jensen, A., Bothtis, D.D. and Sørensen,

C.G. (2012). Simulation modelling for in-field

planning of sequential machinery operations in

cropping systems. Denmark: Aarhus University, PhD

Thesis.

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https://uitm.pure.elsevier.com/en/publications/factors-affecting-farmers-satisfactions-with-mechanized-rice-harv

https://uitm.pure.elsevier.com/en/publications/factors-affecting-farmers-satisfactions-with-mechanized-rice-harv

https://www.researchgate.net/journal/2319-6890_International_Journal_of_Engineering_Research

https://doi.org/10.17950/ijer/v4s1/108

https://www.researchgate.net/journal/1991-6426_World_Applied_Sciences_Journal


 *Corresponding author.

Email: ubonganyi@gmail.com

eISSN: 2550-2166 / © 2020 The Authors. Published by Rynnye Lyan Resources

  Food Research 4 (4) : 982 - 990 (August 2020)
  Journal homepage: http://www.myfoodresearch.com

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Prevalence of Bacillus cereus s.l. in ultra-high temperature chocolate milk from

selected milk manufacturers in Malaysia

1,*Ubong, A., 5New, C.Y., 2Chai, L.C., 3Loo, Y.Y., 1Nor Khaizura, M.A.R., 4Kayali, A.Y.

and 1Son, R.

1Department of Food Science, Faculty of Food Science and Technology, Universiti Putra Malaysia, 43400

UPM Serdang, Selangor, Malaysia.
2Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia.
3Department of Food Science, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600

UKM Bangi, Selangor.
4Department of Global Humanosphere, Center for Southeast Asian Studies, Kyoto University, Kyoto 606-

8501, Japan.
5Go Plus Services Sdn. Bhd., 97A, Jalan BP 6/3, Bandar Bukit Puchong, 47120 Puchong, Selangor Darul

Ehsan, Malaysia

Article history:

Received:  14 December

2019

Received in revised form:  8

February 2020

Accepted:  10 February 2020

Available Online: 8 March

2020

Keywords:

Prevalence,

Bacillus cereus s.l.,

UHT chocolate milk,

MPN-PCR

DOI:
https://doi.org/10.26656/fr.2017.4(4).417

Abstract

Bacillus cereus is a major foodborne pathogen of great concern to the dairy industry

owing to its resilient spores as well as the adverse effect of its toxins. At present, there is

no informational study available to solve or pinpoint the UHT chocolate milk

contamination issue in Malaysia. This work aimed to investigate the prevalence and

contamination level of B. cereus s.l. in UHT chocolate milk and to suggest the appropriate

solution for the issue. In the present study, B. cereus s.l. prevalence and contamination

level in individually packed UHT chocolate milk from processing factories was evaluated.

The prevalence and concentration of B. cereus s.l. were determined via MPN-PCR (Most

Probable Number-Polymerase Chain Reaction) assay. Results showed that 31.11% from

220 of UHT chocolate milk tested contained Bacillus spp.; of this Bacillus spp. positive

samples, 24.30% were also positive for B. cereus s.l. with concentration ranging from less

than 3 to more than 1100 MPN/mL. Findings from this study highlighted the possibility of

UHT chocolate milk as a potential source of B. cereus s.l. infection. Therefore, findings

emphasized the needs to revise, monitor and improve UHT sterilization process to reduce

infection risk. Furthermore, it is also essential to maintain the hygiene to minimize initial

microbial load and contamination of UHT chocolate milk, beginning from production to

retail.

1. Introduction

Bacillus cereus is a Gram-positive, rod-shaped and

spore-forming bacteria which commonly dwells in soil

(Jenson and Moir, 2003; Zhou et al., 2008). Apart from

their natural habitat, they are also widely distributed in

the environment (Reyes et al., 2007). This bacterium was

first defined by Frankland and Frankland (1887) after it

was isolated from air in a cowshed (Larsen and

Jørgensen, 1997).

Often, this bacterium has been considered as fairly

apathogenic; however, B. cereus has also been

implicated in various infections in man (Kotiranta et al.,

2000; McDowell et al., 2019). Apart from that, B. cereus

commonly contaminated proteinaceous food such as

meat, milk and fish, as well as farinaceous food such as

rice, pasta, pastry and noodles (Beecher, 2001;

Messelhäusser et al., 2014). Foodborne illness related to

B. cereus appear as two distinct symptoms; diarrhoeal

and emetic. Diarrhoeal symptoms often characterised as

diarrhoea and abdominal pain, usually eight to 16 hours

after consumption of contaminated food; whereas emetic

symptoms can be observed as nausea and vomiting,

usually one to five hours (Agata et al., 2002; Caroll et

al., 2019). Back in 2013, a massive food poisoning

outbreak related to enterotoxigenic B. cereus was

reported by Schmid et al. (2016). The outbreak was due

to consumption of contaminated mashed potato dishes

containing B. cereus loads of 105 CFU/g. An outbreak

https://orcid.org/0000-0001-5347-5606

983 Ubong et al. / Food Research 4 (4) (2020) 982 - 990

eISSN: 2550-2166 © 2020 The Authors. Published by Rynnye Lyan Resources

involving B. cereus was also reported in Italy. The

analysis found that basmati rice was contaminated with

B. cereus and the bacterium was also found in patients’

stool (Martinelli et al., 2013). Following that, a large

outbreak was reported in 55 nurseries in the United

Kingdom after the consumption of food prepared by the

same company. The microbiological analysis result

showed that B. cereus was detected in dried haricot

beans (Nicholls et al., 2016).

B. cereus is a common contaminant in the food

industry. It was detected in various foods and food

products; among food products that are associated with

B. cereus that have been discovered in other studies are

dried milk products (Reyes et al., 2007), RTE cereals

(Lee et al., 2009; Lesley et al., 2013), rice (Sarrías et al.,

2002; Sandra et al., 2012), minced beef (Samapundo et

al., 2011; Bashir et al., 2013; Lesley et al., 2017; Food

Safety News, 2018), raw milk and pasteurised milk (de

Paula Pacheco-Sanchez and de Massaguer, 2007).

Milk is one of many beverages enjoyed by many

people from all walks of life due to its nutritional value,

with those available in the market are mostly pasteurised

and UHT processed. Processed UHT milk has been

deemed safe for consumption due to the efficient

elimination of microorganisms during UHT processing.

In spite of this, as a common contaminant of raw milk

(Christiansson et al., 1999; Bartoszewicz et al., 2008),

spores of B. cereus can still be detected in the final UHT

products (Vyletělová et al., 2002; Tortora et al. 2016b).

Therefore, consuming UHT milk could increase the risk

of exposure to foodborne pathogens, specifically B.

cereus (Bahk et al., 2007; Abraha et al., 2017).

In Malaysia, UHT milk is a part of the nutritional

program with the aim to reduce malnutrition among

school children especially those in rural areas. A total of

7613 schools were supplied with UHT milk for the

1Malaysia Milk Programme (PS1M) to benefit 1.53

million children (Bernama, 2012). The programme came

to a halt when there was a local outbreak of B. cereus in

UHT milk which had affected 191 students (Yusof,

2011). To the author’s knowledge, there is insufficient

study on the microbiological safety on UHT milk,

especially flavoured milk which is more favoured by the

children.  Therefore, the current study was conducted to

investigate the prevalence and contamination level of B.

cereus s.l in UHT milk. The outcome of the study will

provide an insight into the UHT process of chocolate

milk in Malaysia and identify the possible risk factors.

On the whole, data collected from this study will help to

contribute towards understanding the risk of B. cereus

infection through the consumption of UHT chocolate

milk.

2. Materials and methods

2.1 Sampling

A total of 214 packages of UHT milk were collected

from two manufacturers (A and B – factory names were

not to be disclosed due to confidential purposes), in

Malaysia were analysed in this study. The samples were

transported and immediately analysed upon arrival to the

laboratory.

2.2 Most probable number-polymerase chain reaction

(MPN-PCR)

Enumeration was performed according to the method

described by Lee et al. (2009), and Tallent et al. (2012)

with modifications according to Cappuccino et al. (2014)

and Sandra et al. (2012). Homogenized samples in

tryptic soy broth were diluted to 100-fold and 1000-fold

for MPN three tubes test and incubated at 37°C for 48

hrs. Turbid MPN tubes were subjected to DNA

extraction via boil cell method (Tunung et al. 2010).

Briefly, one mL of culture was centrifuged at 13, 400 × g

for 1 min and the pellet was resuspended in 500 µL TE

(Tris-EDTA) buffer. The suspension was boiled for 10

min and immediately cooled at -20°C for 10 mins. The

suspension was centrifuged again for 1 min at 13, 400 ×

g. The boiled cell lysate was used as template for PCR

assay.

Bacillus spp. and B. cereus s.l. was detected by PCR

assay performed in a 25 µL reaction mixture containing

5 µL of 5× Green GoTaq® Flexi Buffer, 1.5 mM of

MgCl2, 0.08 mM of dNTP mix, 0.2 µM of each primer, 1

U/µL Taq polymerase (Promega, USA), and 2.5 µL of

DNA template. Amplification was performed at 94°C (5

mins) for pre-denaturation, denaturation at 94°C (1 min),

annealing at 55°C (1 min), extension at 72°C (1 min) and

final extension at 72°C (4 mins). The products were

electrophoresed on 1% agarose gel with 1× TBE (Tris-

Borate-EDTA) buffer at 100 V for 28 mins. Table 1

shows the list of primer sequences used in this study.

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Primer Sequence Annealing Temperature Reference Amplicon Size (bp)

Bacillus
spp.

F: 5’-TCACCAAGGCAACGATGCG-3’
55 °C, 1 min Wu et al. (2005) 1114

R: 5’-CGTATTCACCGCGGCATG-3’

B. cereus
F: 5’-GAGTTAGAGAACGGTATTTATGCTGC-3’

55 °C, 1 min
Schraft and

Griffiths (1995)
411

R: 5’-CTACTGCCGCTCCATGAATCC-3’

Table 1. Primer sequences used in this study.

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2.3 Isolation of B. cereus isolates

Turbid MPN tubes were streaked on Mannitol-Yolk-

Polymyxin agar and incubated at 37°C for 18 to 24 hrs.

Suspected single pink to purple colonies which were

surrounded by a ring of dense precipitation of B. cereus

was picked for further confirmation by PCR.

3. Results and discussion

In this study, Bacillus spp. and B. cereus s.l. were

detected in 31.11% (67/214) and 24.30% (52/214) of the

UHT chocolate milk samples, respectively, with MPN

distribution ranging from less than 3 to more than 1100

MPN/mL. Majority of the samples were detected with

concentration less than 3 MPN/mL; 68.69% and 75.70%

for Bacillus spp. and B. cereus s.l. respectively. For

concentration range of 3 to 9.4, 11 to 93, and 120 to

1100 MPN/mL for Bacillus spp., the percentage was

19.61%, 5.61% and 0.47% respectively; for B. cereus

s.l., the percentage were 14.49%, 3.74% and 1.87%

respectively. Bacillus spp. and B. cereus s.l. with

concentration more than 1100 MPN/mL account for

6.07% and 4.21% of the samples respectively (Figure 1).

Figures 2 and 3 show representative positive samples

detected with Bacillus spp. and B. cereus s.l.

The median MPN distribution samples collected in

this study is less than 3 MPN/mL, in which it accounts

for the majority of the MPN distribution. Thence, it is

suggested that majority of the chocolate milk samples

were safe for consumption as B. cereus s.l. was not

detected in any of the samples. However, it should be

kept in mind that when samples were ‘not detected’ with

B.  cereus s.l., it does not mean that the samples were

free from the said bacterium. The number of B. cereus

s.l. was below detected limit, therefore, they were not

detected by MPN-PCR assay. Nevertheless, samples

with less than 3 MPN/mL could also pose a threat to

consumers.

The results of the study were in agreement with

Lesley et al. (2017) who reported 30% of UHT milk

sampled in retail markets in Sarawak, East Malaysia.

Vidal-Martins et al. (2005) detected B. cereus in 11.8%

of the 110 samples of UHT milk in their research,

wherein the detection is lower compared to the present

study. A slightly higher detection was described by

Rezende-Lago et al. (2007); 58.3% of 120 samples

consisting of raw milk, milk powder, UHT milk and

pasteurised milk were contaminated with B. cereus, but

detection was lower in UHT milk (13.3%). The presence

of B. cereus in UHT milk was also reported in previous

studies (Bartoszewicz et al., 2008; Ghellai and

Moussaboudjemaa, 2013) which are parallel with the

present study. Prevalence of B. cereus s.l. in this study is

higher compared to the prevalence of B. cereus in UHT

milk mentioned previously in other studies. It can be

suggested that it might be due to the high microbial load

from raw materials which can lead to the high number of

successive spores (Vyletělová et al., 2002; Amor et al.,

2018).

In another related study, Schlegelova et al. (2003)

reported that 31.0% of dairy foodstuff samples were

contaminated with B. cereus, showing similar prevalence

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Figure 1. Percentage of contamination level of Bacillus spp.

(indicated in blue bars) and B. cereus (indicated in red bars) in

UHT chocolate milk.

Figure 2. Representative amplification of 16S rRNA gene for

the detection of Bacillus spp. (1114 bp). Lane M: 1 kb DNA

ladder (Promega, USA); lane 1 to 3: representative positive

samples at 1114 bp; lane 4: positive control (B. cereus ATCC

33019); lane 5: non-Bacillus spp. sample.

Figure 3. Representative amplification of cereolysin AB gene

for the detection of B. cereus s.l. (411 bp). Lane M: 100 bp

DNA ladder (Promega, USA); lane 1 to lane 3: representative

positive samples at 411 bp; lane 4: positive control (B. cereus

ATCC 33019); lane 5 and lane 6: non-B. cereus sample.

985 Ubong et al. / Food Research 4 (4) (2020) 982 - 990

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to the present study. Another related research stated that

B. cereus was found in 45.9% of dried milk products

(Reyes et al., 2007) and analysis by a local study

indicated that 78% of 111 RTE cereals were detected

with B. cereus s.l., showing a higher detection compared

to present study (Lee at al., 2009). Apart from that, more

information on B. cereus isolation from various milk and

milk products has also been described from the following

researches; fresh milk as well as heat-treated milk such

as pasteurised milk and UHT milk (Vyletělová et al.,

2002; Salustino et al., 2009; Hassan et al., 2010; Fallah

et al., 2011; Montanhini et al., 2013; Kumari and Sarkar,

2016).

UHT processing for milk is a process of continuous

heating at temperatures higher than 130°C (generally at

140°C to 150°C) for a holding time of a few seconds

(generally 2 to 10 s). Following that, the milk is

aseptically packaged to produce a ‘commercially sterile’

product. Hence, under these conditions, microorganisms

that are capable of growing under normal condition of

product storage are eventually destroyed (Datta et al.,

2002; Kjaerulff, 2013). In food industries, UHT

processing is the favoured way of sterilising chocolate

flavoured milk as it will enhance the flavour of the

chocolate without causing bitter aftertaste. What is more,

it provides an overall smoothness and favourable taste

and at the same time, it extends its shelf-life (Bixler et

al., 2001; Tiwari and Asgar; 2017). Although the

chocolate milk has initially gone through the sterilisation

process before being distributed to local retail markets,

there were many possible aspects to be considered for B.

cereus s.l. recontamination on the final products.

Doyle et al. (2016) described that the possible reason

for contamination of milk occurs during the outdoor

grazing period. It was suggested that soil was the main

source of contamination for milk by means of cow’s teat

that came in contact with soil (Christiansson et al., 1999;

Doyle et al., 2016). On that account, the spores

eventually came in contact with raw milk during the

cows’ milking process. Spores in manure may also

contribute to some degree of spore in milk, given with

high spore content in the soil. In the processing

environment, B. cereus spores in raw milk will continue

to persist after pasteurisation and later on will continue

to colonise pipes, storage tanks and filling machines

(Jenson and Moir, 2003; Gopal et al., 2015). However,

most milk manufacturing plants in Malaysia used

reconstituted milk for economic reasons. The spores

present in raw milk could remain even after the milk was

transformed into a powdered form may suggest highly

thermo liable spores which are greatly influenced by the

genetic group (Sandra et al., 2012; Lesley et al., 2013).

The spores transfer was suspected to be likely

initiated from the raw materials. Commercially produced

chocolate milk is usually prepared by mixing raw

ingredients such as raw milk or milk powder with cocoa

powder, sugar, thickening agent, food stabiliser and

preservatives. Selected raw ingredients collected from

the factories were subjected to examination for the

presence of B. cereus s.l. From 32 samples of raw

ingredients, 22 (68.6%) samples were detected with B.

cereus s.l. with MPN distribution ranges from less than 3

MPN/mL to more than 1100 MPN/mL. This indicates

that most of the raw materials contained B. cereus

spores. The spores which might be present in these raw

ingredients may successfully be transferred during the

milk processing course (Vyletělová et al., 2002; Amor et

al., 2018) and surviving the UHT process, if the spores

are incompletely inactivated. Generally, B. cereus

utilises glucose as its main carbon source (Tallent et al.,

2012). Apart from that, it is also capable of fermenting

sucrose (Aryal, 2019). The high content of sugar in UHT

chocolate milk (7.2 g of sucrose per 200 mL of UHT

chocolate milk pack) could support the spore

germination and growth. In addition, the use of different

food ingredients, especially exotic and imported, creates

new niches and risk of introducing new unknown heat-

stable spore-formers into the production process

(Witthuhn et al., 2011). According to a study conducted

by Witthuhn et al. (2011) on the thermal stability of

spores, it was concluded that cocoa powder was shown

to contain highly heat-resistant spores.

The initial designs of UHT processes focused on the

inactivation of thermophilic bacterial spores such as

spores of Geobacillus stearothermophilus due to their

survivability in UHT heating (Witthuhn et al., 2011). It

was reported that the spores are activated at 121°C and

to inactivate the spores requires temperatures well above

121°C (Russell, 1982; Huesca-Espitia et al., 2016).

However, the introduction of extremely heat-resistant

mesophilic spore-former Bacillus sporothermodurans

(IDF, 2000) had impacted the UHT industry, making a

fine line in achieving the inactivation required with the

milk quality. In reality, higher temperature will ensure

complete inactivation of the spores. Despite the proposed

inactivation kinetics, severe chemical changes will be

observed, for example, browning, loss vitamins, fouling

and gelation, which degrades the quality of milk. What

more when the distribution of unknown very heat-stable

spores or the evolution of spore-formers such as B.

cereus, a mesophilic and foodborne pathogen into

producing heat-stable spores.

During the production of UHT milk, there is a

possibility for the activation of the spores which led to

germination. Levinson and Hyatt (1970) reported that the

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activation of B. cereus spores is within 62°C to 78°C and

maximal rate of spore germination was at 64°C to 68°C.

The activation temperature is within the range of the

holding temperature of most UHT processes which

brings the possibility that the spore’s germination in a

longer indirect heating system, posing a hazard to the

design of safe processes.

Apart from their ability to survive harsh environment

via endospores, B. cereus s.l. is also known to form

biofilm which is a huge concern for the food industry. In

fact, B. cereus’ ability to form biofilm has been

extensively reported in many researches, colonising

surfaces such as stainless steel, glass, plastic, leafy

vegetable and polystyrene (Ryu and Beuchat, 2005;

Hsueh et al., 2006; Houry et al., 2009; Elhairy, 2011;

Majed et al., 2016; Hussain et al., 2018). It is possible

that during processing, B. cereus cells anchor to the

surfaces of pipe, storage tanks and filling machines,

eventually colonising them (Jenson and Moir, 2003;

Gopal et al., 2015; Majed et al., 2016). They will

ultimately produce matrix and form stronger biomasses,

with a matrix forming a hydrophobic envelope

surrounding the biofilm. The extracellular matrix is

primarily comprised of polysaccharides, such as

cellulose, proteins or exogenous DNA. The matrix can

fix to hard surfaces such as food industry equipment,

transport, dispensing and storage surfaces and soil; or to

biological structures such as vegetables, meat, bones and

fruits. The embedded cells are protected against toxic

compounds, due to the matrix’s structural role which

responsible for durability and persistency of biofilm.

Biofilm formation provides many advantages to

microbial cells, especially in the food industry setting.

For example, physical resistance against desiccation,

mechanical resistance against liquid streams in pipelines,

and chemical protection against chemicals,

antimicrobials and disinfectants used in the food industry

(Flemming et al., 2016; Galié et al., 2018). It can contain

spores as well as vegetative cells with spores and result

in recontamination during the detachment of these

biofilms (Faille et al., 2014).

Thenceforth, biofilms are very difficult to eliminate

as they exhibit resistant to high temperature and

disinfection processes compared to bacteria in the free-

living environment. In sync with spores, it gives B.

cereus high resistance to numerous stress as well as high

adhesive capacity on various substrates, which includes

stainless steel, a common material used in food

processing. Moreover, sporulation occurs at a very high

level in biofilms, which makes them difficult to

eliminate. It is of great concern when biofilms start to

form in areas which are difficult to clean, for example,

crevices, valve, gaskets and dead ends (Donlan, 2002;

Majed et al., 2016).

Contamination of UHT chocolate milk could also

take place during the cooling process as milk was passed

through the cooling chamber, a potential area for biofilm

colonisation. Contamination also causes by the

reintroduction of spores during the filling process; when

chocolate milk was dispensed into smaller packs. These

spores might come from biofilms that have colonised

dispenser nozzles and eventually released into previously

sterilised chocolate milk. Another possible route of

contamination was the chocolate milk could also expose

to airborne B. cereus spores during the filling process

(Actor, 2012). Hence, when optimum temperature is

reached for B. cereus growth, the spores will germinate

and return to their vegetative state (Tortora et al., 2016;

Majed et al., 2016).

It is critical to take note that low numbers of B.

cereus cells could also cause illness. Lee et al. (2009)

mentioned in their study on the incident of a low

infective dose of B. cereus that caused serious food

poisoning outbreak in Norway; with a number as low as

103 to 104 cells/mL, food industries should be a concern

(Granum, 1994). A total of 4.21% of UHT milk samples

from this study contained more than 1100 MPN/mL of B.

cereus. Considering the fact that 103 cells/mL could lead

to infection, this UHT milk could pose risk to

consumers. In addition to that, milk is a good medium to

support the germination of B. cereus due to its nutritious

content (Ray and Bhunia, 2008). As such milk is

consumed more by infant and children for its nutrients

and who bear the highest foodborne disease burden (Kirk

et al., 2015), it is, therefore, necessary to monitor the

microbiological safety of milk.

4. Conclusion

Detection of B. cereus s.l. and other foodborne

pathogens is vital for the safety of food products.

Findings from this study highlighted the possibility of

UHT chocolate milk as a potential source of B. cereus

s.l. infection. In this manner, it is of significant important

to revise, monitor and improve UHT sterilization and

packaging processes in order to reduce infection risk.

From food safety point of view, thorough and continuous

monitoring of B. cereus s.l. during UHT milk production

process is important in order to address B. cereus s.l.

infection issue as UHT milk is a potential medium to

acquire its infection. Furthermore, it is also imperative to

supervise and maintain the hygienic level to minimize

initial microbial load and contamination, beginning from

production to table in order to safeguard consumers

along with public health.

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Conflict of Interest

The authors declare no conflict of interest.

Acknowledgment

This research was supported by the National

Biotechnology Division, Ministry of Science,

Technology Malaysia under the grant (02-01-04-

SF1526) and Fundamental Research Grant Scheme

(FRGS) of Ministry of Higher Education (MOHE),

Malaysia (01-01-18-2015FR) in parts with AMED under

Grant No.: JP15km0908001.

The authors acknowledged the late Professor Dr

Mitsuaki Nishibuchi from the Department of

Humanosphere, Center for Southeast Asian Studies,

Kyoto University, Kyoto, Japan, for his generous support

in this research.

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