 *Corresponding author.

Email: p_atp@hotmail.com

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

  Food Research 3 (2) : 138 - 144 (April 2019)
  Journal homepage: http://www.myfoodresearch.com

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Development of gluten-free cream puff with the addition of

carboxymethylcellulose and carrageenan

1*Penjumras P., 1Janmeesup, C., 1Umnat, S., 1Chokeprasert, P., 1Wattananapakasem, I. and
2Phaiphan, A.

1Program of Food Science and Technology, Maejo University-Phrae Campus, 54140 Rongkwang, Phrae,

Thailand.
2Program of Food Science and Technology, Faculty of Agriculture Ubon Ratchathani Rajabhat University,

34000 Ubon Ratchathani, Thailand.

Article history:

Received: 9 July 2018

Received in revised form: 21

October 2018

Accepted: 24 October 2018

Available Online: 6

November 2018

Keywords:

Gluten-free,

Rice flour,

Bakery product,

Hydrocolloid

DOI:
https://doi/org/10.26656/fr.2017.3(2).133

Abstract

The consumption of gluten-free products is gaining attention since a growing number of

people who suffer from celiac disease. However, many gluten-free products currently

available in market are of low quality. The objective of this study was to evaluate the

effect of the addition of carboxymethylcellulose and carrageenan on physical properties

and sensory characteristics of cream puff made with 100% red brown jasmine rice flour.

Cream puffs using 1% hydrocolloid at different concentrations of CMC mixed with

carrageenan (0.25%+0.75%, 0.50%+0.50%, and 0.75%+0.25%) were developed and

compared to a control sample prepared from wheat flour. The results showed that the use

of rice flour reduced the effects of all physical properties and sensory characteristics.

Compared to control, cream puff made with rice flour had a lower specific volume and

darker. However, the incorporation of 0.50% CMC mixed with 0.50% carrageenan

presented quality attributes similar to that of the control sample due to hydrocolloids act as

polymeric substances that should mimic the viscoelastic of gluten. Therefore, it is feasible

for gluten-free cream puff application.

1. Introduction

Among cereals, wheat has specific proteins that

make it ideal for certain applications. The unique

properties of wheat are generally ascribed to the visco-

elastic properties of its gluten proteins. While

monomeric gluten proteins (gliadin) show viscous

behavior, polymeric gluten proteins (glutenin) are elastic

(Veraverbeke and Delcour, 2002). Thus, wheat gliadins

and glutenins, in the presence of water and mechanical

work, form a continuous phase named gluten network. It

is responsible for the extensible and cohesive properties

of the dough while reducing its stickiness. Wheat dough

is characterized for its tenacity, dough resistance to

stretching and elasticity, dough ability to regain its

original shape after being stretched. The unique elasticity

of glutenin results to a large extent from its polymeric

nature. Glutenin is a highly heterogeneous mixture of

polymers consisting of a number of different high- and

low-molecular-weight glutenin subunits linked by

disulfide bonds (Veraverbeke and Delcour, 2002; Gómez

and Sciarini, 2015). A variety of cereal foods are

produced from wheat flour, such as bread, cake and

pasta, all of which, contain gluten. However, many

people suffer from gluten-related disorders. Celiac

disease, also known as gluten intolerance to certain

amino acid sequences found in the prolamin fraction of

wheat (gliadin), rye (secalin) and barley (hordein)

(Itthivadhanapong et al., 2016). Gluten replacement is

one of the most challenging issues for food science and

technology since finding high-quality gluten-free foods is

a major issue for celiac disease sufferers (Turabi et al.,

2010; Itthivadhanapong et al., 2016). Among gluten-free

raw materials that can be used for this purpose, the most

important ones are cereal flours without gluten, as well

as native starches. Rice flour is one of the most suitable

raw materials due to its hypoallergenic properties, low

sodium content, bland taste, and its easy availability in

the market (Torbica et al., 2012). Nowadays, the

consumption of pigmented rice such as brown, red,

purple or black pericarp has gain attention in some Asian

countries (Jeng et al., 2012). Pigmented rice is also

known as a source of antioxidant compounds including

flavonoid, anthocyanin, phytic acid, proanthocyanidin,

tocopherols, tocotrienols, γ-oryzanol, and phenolic

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compounds (Butsat and Siriamornpun, 2010; Goufo and

Trindade, 2014; Thitipramote et al., 2016). Thitipramote

et al. (2016) reported that two native Thai pigmented rice

(Brown red jasmine rice and Kam leumpua; black rice)

compose of the higher phenolic compounds and greater

antioxidant activities than Japanese brown rice. The

greatest amount of these compounds and activities was

found in red rice (Brown red jasmine rice). The

phytochemical in pigmented rice, such as phenolics and

anthocyanins are related with reducing the risk of

developing chronic diseases, such as diabetes, obesity,

cancer, and cardiovascular disease (Mau et al., 2017).

Some bakery products have been studied on the use of

rice flour as a replacement for wheat flour, such as rice

flour cake containing lupin flour and whole buckwheat

flour (Levent and Bilgiçli, 2011); chiffon cake prepared

with black rice (Mau et al, 2017); yellow cake prepared

from rice flour and peanut paste (Jaganathan, 2016), and

white bread made from rice flour mixed with cornstarch

and cassava starch (López et al., 2004). However, the

use of rice flour to prepare cream puff has not been

exploring yet. Thus, it would be beneficial to develop a

novel formula for cream puff made from red jasmine

rice. The replacement of gluten presents a major

technological challenge, as it is an essential structure-

building protein. To tackle this problem, hydrocolloids

were incorporated in gluten-free flour to mimic the

viscoelastic properties of gluten (Lazaridou et al., 2007;

Pahwa et al., 2016). The addition of hydrocolloids in

gluten-free formulation products is necessary in order for

the hydrocolloids to act as polymeric substances that

should mimic the viscoelastic properties of gluten and

increase the dough’s gas-retaining ability (Sabanis and

Tzia, 2011). The hydrocolloid reduced the availability of

water for granule swelling. The increased viscosity

thereby increased the shear forces exerted on the swollen

granules, thus increasing the breakdown viscosity.

Associations between starch polymer molecules and

hydrocolloid molecules could be responsible for the

increase in the setback and final viscosity. Hydrocolloid

molecules bound water reduced the mobility of the starch

chains and thereby retard retrogradation (Satrapai and

Suphantharika, 2007). Therefore, the purpose of this

study was to develop gluten-free cream puff with the

addition of hydrocolloids including

carboxymethylcellulose and carrageenan.

2. Materials and methods

2.1 Materials

Commercial red brown jasmine rice was purchased

from the local market in Phrae province, Thailand

(Kaijae brand, Thailand). All-purpose wheat flour (Kite

brand, UFM Food Center Co., Ltd, Thailand), cake flour

(Red Lotus brand, UFM Food Center Co., Ltd,

Thailand), butter (Allowri brand), margarine (Best

Foods, Thailand) and fresh whole eggs (~50 g/piece)

were used in this study, carboxymethylcellulose (CMC)

and carrageenan were purchased from Union Science

Co., Ltd. Thailand.

Red jasmine rice was ground using blender (Model

HR 2115, Philips, Indonesia), and screened through a

150-mesh sieve five times. The rice powder was

contained in an airtight box and then kept at room

temperature (~25°C) before use.

2.2 Analysis of chemical composition of wheat flour and

rice flour

Moisture content (oven at 105°C), Ash content

(muffle furnace at 550-600°C), lipids (Soxhlet method),

proteins (Kjeldahl method) and fiber were evaluated

according to the official methods of the AOAC (2012).

Total carbohydrates were calculated following equation

(1).

2.3 Preparation of cream puff

The basic cream puff recipe was chosen according to

our preliminary study. The control cream puff

formulation contained the following ingredients: 120 g

all-purpose wheat flour, 120 cake flour, 480 g water, 75

g butter, 75 g margarine and 350 g fresh whole egg. For

red jasmine rice flour cream puff, 100% rice flour (240 g

rice flour) was used to substitute wheat flour. CMC and

carrageenan were added in 1% of flour basis. Cream

puffs using 1% hydrocolloid at different concentrations

of CMC mixed with carrageenan (0.25%+0.75%, 0.50%

+0.50%, and 0.75%+0.25%). The cream puffs made

from rice flour without hydrocolloid were also prepared.

The cream puffs were manufactured using the following

steps: flour was sieved through a 150-mesh sifter. Butter

and margarine were then mixed in boiled water and

stirred vigorously over low heat until mixture left the

pan. The mixture was removed from heat and let it cool

for 5 mins. Hydrocolloid was dissolved in cold water and

blended with the mixture. Eggs were beaten with the

mixture until smooth and velvety. The mixture was

dropped onto a baking tray and baked in preheated oven

200°C for 20 mins. Cream puff was removed from the

oven, cooled for 1 hr and kept in an airtight box and put

in freezer at 8°C until analysis.

2.4 Physical characteristics of batters

The viscosity of batter was performed with a

viscometer (RVT, Brookfield viscometer, USA). The

400 mL of cream puff batter was poured into a 500 mL

beaker and its viscosity was measured. The specific

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gravity of batter was determined by dividing the weight

of a standard container filled with batter by the same

container filled with water (Lu et al., 2010). Each

analysis was carried out in triplicates.

2.5 Physical characteristics of cream puffs

The physical characteristics of cream puff, including

specific volume, color, weight loss and moisture content.

The specific volume (mL.g-1) of the cream puff batter

was obtained by the ratio between the apparent volume

(mL) and the cream puff mass (g) after baking. The mass

was obtained by weighing the cream puff on a precision

scale (Ohaus, Pioneer PA214, USA). The apparent

volume of the cream puff was determined by the method

of displacement of sesame seeds (Mau et al., 2017). The

color of the cream puffs was measured using a

colorimeter (Color Flex 500, Hunter Lab, USA). Results

were expressed in CIE L* a* and b* values. The cream

puffs were weighted before baking (W0) and after baking

and 1-hour cooling (W1). The weight loss was calculated

using equation (2) (Matos et al., 2014). Moisture content

was measured according to the official methods of the

AOAC (2012).

2.6 Sensory evaluation

The degree of overall preference for baked cream

puffs was determined by hedonic scale test (Mau et al.,

2017). The untrained panelists were recruited from the

students and staff at Maejo University-Phrae Campus.

All untrained panelists were informed on how to evaluate

cream puff. The 50 panelists received 5 samples and

were asked to rate them based on the degree of

preference on a 7-point hedonic scale (1 = dislike

extremely, 4 = neither like nor dislike, and 7 = like

extremely). Panelists evaluated the samples in a testing

area and were instructed to rinse their mouths with water

between samples to minimize any residual effect.

2.7 Statistical analysis

Data were subjected to Analysis of Variance

(ANOVA) using SPSS for Window version 24. In case

of any differences in mean, multiple comparisons were

performed using Duncan’s Multiple Range Test (DMRT)

at 5 % level of significance (P0.05).

3. Results and discussion

3.1 Chemical composition of raw materials

The results for the chemical composition of the

wheat flours and red jasmine rice flour are presented in

Table 1. The composition of all-purpose flour on a wet

basis was 11.73% moisture, 0.49% ash, 0.77% fat,

7.68% protein, and 79.34% carbohydrates. The similar

composition was found in cake flour, but lower in

protein content with 7.08% then resulting higher of

carbohydrates. Meanwhile, the composition of rice flour

was 10.49% moisture, 0.49% ash, 1.88% fat, 6.16%

protein, and 80.98% carbohydrates.

The results indicated that rice flour had lower in

protein content compared to wheat flour (all-purpose and

cake flour) but higher in lipid and fiber. The results

showed different composition with previous work carried

out by Vallejos et al. (2015). They reported the chemical

composition of the rice flour on a dry basis was 7.52%

protein, 0.41% fat, 0.31% ash, and 91.76%

carbohydrates. This may be related to this recent study

reported on a wet basis.

3.2 Physical characteristics of batters

The foremost reason behind the ample use of

hydrocolloids in food is their ability to modify the

rheology of the food system, namely flow behavior

(viscosity) (Saha and Bhattacharya, 2010). The effect of

the addition of hydrocolloids on physical characteristics

of the batters after complete substitution of the wheat

flour by red jasmine rice flour is presented in Table 2.

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(2)

Samples
Chemical compositions (%)

Moisture ns Ash ns Lipid Protein Fiber Carbohydrates

All-purpose flour 11.73±0.52 0.49±0.01 0.77±0.01b 7.68±0.01a 0.71±0.15b 79.34±0.01b

Cake flour 11.74±0.80 0.49±0.00 0.77±0.00b 7.08±0.00a 0.56±0.11b 79.92±0.00b

Rice flour 10.49±0.48 0.49±0.01 1.88±0.42a 6.16±0.42b 2.08±0.21a 80.98±0.42a

Table 1. Chemical composition of wheat flour and rice flour

*Values followed by the different letter within the same column are significantly different from each other (p0.05).

** Values followed by ns within the same column are not significantly different from each other (p>0.05).

Batter samples Viscosity (cP)ns Specific gravity

Wheat flour 20,000±0 1.10±0.05a

Rice flour (CMC: carrageenan)

0:0 18,533±461 0.89±0.02c

0.25:0.75 19,266±702 1.07±0.01a

0.50:0.50 19,667±577 1.08±0.01a

0.75:0.25 20,000±0 1.04±0.00b

Table 2. Physical characteristics of cream puff batter

*Values followed by the different letter within the same col-

umn are significantly different from each other (p0.05).

** Values followed by ns within the same column are not

significantly different from each other (p>0.05).

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The specific gravity of batters estimates the air

incorporated into a batter, a lower specific gravity

indicates of a batter with more air and viscosity. All

batter had a specific gravity higher than 0.75 indicating a

stable emulsion (Lin et al., 1994; Singh, 2012). The

viscosity of all cream puff batter did not differ

significantly with replacing rice flour instead of wheat

flour. The similar result was found by Mau et al. (2017)

who used black rice flour as a replacement for wheat

flour in chiffon cake. However, the viscosity of batter

prepared from rice flour increased slightly with the

addition of hydrocolloids, especially the increase of

CMC ratio. Sidhu and Bawa (2000) reported that CMC

involve its interaction with water.

Carboxymethylcellulose interacts with protein molecules

to provide suspension and solution stability. They found

that the increase of CMC affected the increase of water

absorption in bread batter. A decrease in the specific

volume of batters was observed with the substitution of

the wheat flour by red jasmine rice flour. This could be

related the higher in lipid content as shown in Table 1

then resulting in lower specific gravity.

3.3 Physical characteristics of cream puffs

The effect of the addition of hydrocolloids on

physical characteristics of cream puffs was investigated.

The results are presented in Table 3.

From Table 3, the specific volume, weight loss,

moisture and color of rice flour cream puff differed

significantly compared to wheat flour cream puff. The

cream puff prepared from wheat flour showed higher

specific volume. This is related to higher weight loss

then resulting in lower moisture content and resulting

thin crust as shown in Figure 1. Cream puff structure

setting must be timed so that the air bubble can properly

be expanded by water vapor before it sets. Thus, the

resulting puff structure is highly aerated and has a more

defined structure. In this study, there was no difference

among viscosity of all formula puff batter. However, the

gluten of batter decreased when wheat flour was replaced

with rice flour, and thus, the ability to retain air was

poorer which produced the undesirable aerated structure

with lower volume (Mau et al., 2017). Basically, the

addition of hydrocolloids in gluten-free formulation

products is necessary in order to act as polymeric

substances that can mimic the viscoelastic properties of

gluten and increase the dough’s gas-retaining ability

(Sabanis and Tzia, 2011). However, this recent study

demonstrated that the addition of hydrocolloids did not

increase the dough’s gas-retaining ability but increase

water absorption. It can be seen from higher moisture

content in the final product. From Table 3, the result

summarized that rice flour puff without the addition of

hydrocolloid provided better physical characteristics than

the addition of hydrocolloids. However, this study found

that the rice puff’s structure without the addition of

hydrocolloid had weak crust structure during storage.

The Hunter, L*, a* and b*, values correspond to

lightness, redness, and yellowness, respectively. The

crust color of baked puff was greatly affected by

replacing wheat flour with rice flour as shown in Table

3. The L* and b* values decrease but a* value increased,

indicating that a darker, redder, and less yellow was

obtained as a result of red jasmine rice replacement. The

colors of pigmented rice as red jasmine rice attributes to

anthocyanins and proanthocyanin which present the red

or purple color (Min et al., 2011).

3.4 Sensory characteristics

Sensory characteristics were tested to evaluate

acceptance by the consumer as shown in Table 4.

From Table 4, wheat flour cream puff demonstrated

appearance, color, flavor, taste, texture and overall

acceptance scores of 5.76, 6.10, 5.40, 4.68, 5.42 and

5.04, respectively. Meanwhile, rice flour cream puff

showed appearance, color, flavor, taste, texture and

overall acceptance scores in the range of 4.98-5.04, 5.24-

5.46, 4.64-4.92, 4.00-4.36, 4.58-5.02 and 4.34-4.54,

respectively. Although the substitution of wheat flour

with red jasmine rice flour led to significantly decreased

sensory characteristics the decrease of the score for all

characteristics was less than 1 scale. In addition, all

sensory characteristics of rice cream puff had scored

higher than 4. This means that panelists accepted cream

puff made from rice flour. When compared between

baked rice flour cream puff, there was no difference

among the appearance, color, flavor and overall

acceptance score for all cream puff. However, the taste

score of rice flour cream puff adding CMC: carrageenan

with 0.50:0.50 did not differ significantly compared to

wheat flour cream puff. Moreover, rice flour cream puff

adding CMC: carrageenan with 0.50:0.50 had the highest

score of color, flavor, taste and texture compared to other

rice flour cream puff. In addition, it provided the highest

specific volume as shown in Table 3. Therefore, the

optimum red jasmine rice flour cream puff was the

addition of CMC: carrageenan with 0.50:0.50.

4. Conclusion

The addition of hydrocolloids significantly affected

some characteristics of rice flour cream puff. The ratio

CMC: carrageenan with 0.50:0.50 increased cream puff’s

specific volume and had highest sensory score. The

sensory qualities of each characteristic were 4.38-5.46 on

a 7-point hedonic scale, indicated that the cream puff

was moderately acceptable. Therefore, red jasmine rice

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flour cream puff could be developed as an alternative

bakery product for consumers who suffer from celiac

disease and a novel functional food with antioxidant

properties due to rich in bioactive compound.

Acknowledgment

The authors would like to acknowledge Maejo

University-Phrae Campus for laboratory equipment

support.

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Weight loss

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Moisture

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  Color

L* a* b*

Wheat flour 5.11±0.19a 26.16±1.78a 8.08±0.30a 58.82±0.29a 4.56±0.11e 39.60±0.12a

Rice flour (CMC: carrageenan)

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0.75:0.25 3.31±0.27c 21.15±2.39c 8.75±0.31b 38.91±0.32d 10.04±0.33a 34.09±0.71c

Figure 1. Photograph of cream puffs (a) wheat flour cream puff; (b) wheat flour cream puff crust; (c) rice flour cream puff with-

out hydrocolloid; (d) rice flour cream puff crust without hydrocolloid

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Table 3. Physical characteristics of baked cream puffs

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0.25:0.75 4.98±1.27b 5.38±1.03b 4.84±1.53b 4.28±1.77b 4.80±1.54bc 4.54±1.56b

0.50:0.50 5.00±1.34b 5.46±1.13b 4.92±1.43b 4.36±1.72ab 5.02±1.44b 4.38±1.52b

0.75:0.25 5.04±1.24b 5.24±1.09b 4.76±1.36b 4.00±1.55b 4.58±1.54c 4.52±1.40b

*Values followed by the different letter within the same column are significantly different from each other (p≤0.05).

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 *Corresponding author.

Email: kkanta_36@stamforduniversity.edu.bd

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

  Food Research 4 (4) : 1312 - 1316 (August 2020)
  Journal homepage: http://www.myfoodresearch.com

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Isolation of pathogenic microorganisms and determination of their antibiotic

resistance patterns collected from different bakery products of Dhaka city

*Das, K.K., Sarkar, A. and Hossain, A.

Department of Microbiology, Stamford University Bangladesh, 51 Siddeswari Road, Dhaka 1217,

Bangladesh

Article history:

Received: 28 November 2019

Received in revised form: 14

January 2020

Accepted:  23 January 2020

Available Online: 22

February 2020

Keywords:

Bakery products,

Microbiological analyses,

Antibiotic resistance,

Food quality

DOI:
https://doi.org/10.26656/fr.2017.4(4).400

Abstract

Bakery products play an important part in the diet of daily life and provide vital nutrients

to human. But pathogenic microorganisms may get entry to bakery items through

improper handling and storage which may lead some disease to consumers. For this

reason, the objective of this study was to detect pathogenic bacteria in different bakery

products collected from different places of Dhaka city. In this study, commonly consumed

bakery items such as sweets, biscuit, cake and bread samples were collected from different

locations for the detection of pathogenic microorganism. Here, all the samples were found

to be contaminated huge microbial count up to 106 CFU/g. Klebsiella spp. and

Pseudomonas spp. were most predominate among all. On the other hand, Escherichia coli

was totally absent in all the samples. This result indicated that bakery products prepared

under unhygienic environments and which may serve as a reservoir of various pathogenic

bacteria and most of them were showed resistance against common antibiotics. To ensure

the health safety of consumer’s government should take necessary actions to educated

food handlers.

1. Introduction

Bakery products are most consumed food worldwide

and a good source of different essential nutrients such as

carbohydrates, proteins, lipids, vitamins and minerals

(Potter and Hotchkiss 2006; Saranraj and Geetha, 2012).

As they contain low water activity and cooked under

high temperature freshly baked products are free from

microbes and do not contain pathogenic microorganisms

therefore from the microbiological point of view bakery

products are generally considered as safe food (ICMSF

2002; Saddozai and Khalil, 2009; Saranraj and Geetha

2012; Khanom et al., 2016). Cooking method, lower pH,

the addition of preservative and low moisture in bakery

items can prevent products from bacterial spoilage and

also extended their self-life (Berghofer et al., 2003).

But improper storage conditions, unhygienic

handling may support the proliferation of various

microorganisms in cereal grains and bakery products

(Deibel and Swanson, 2001). Some common bacteria

like Bacillus sp. and mold include Rhizopus sp., Mucor

sp., Penicillium sp., Eurotium sp., Aspergillus sp. and

Monilia sp. are responsible for spoilage which depends

on seasons, type of products and methods of processing

(Seiler, 2000; Saranraj and Geetha 2012). Several studies

conducted in various countries have reported that food

poisoning resulting from contaminated flour (Saddozai

and Khalil, 2009; Saranraj and Geetha 2012; Khanom et

al., 2016). Butothers research on wheat and flour were

reported that they contain pathogenic microorganisms

like Salmonella spp., Escherichia coli, Bacillus cereus

and other spoilage micro-organisms in low levels

(Richter et al., 1993; Victor et al., 2013). So, it would be

different physical, chemical and microbial factor-like

slicing equipment, post-baking contamination from the

handler, bread coolers, conveyor belts, racks and high

moisture can also influence the spoilage of bakery

products (Saddozai and Khalil, 2009; Saranraj and

Geetha 2012). Bakery products come in contact with

moulds, yeasts and bacteria such as the rope-causing heat

- resistant endospore-forming Bacillus subtilis during

production and inappropriate storage (Earle and Putt,

1984). Mold spores are killed in the baking process

(Knight and Menlove, 1961), leaving often

contamination to be the source of spoilage problems.

Penicillium is the most common fungal contaminants

which also necessary for sourdough bread (Legan and

Voysey, 1991). Some bacterial spore-like Bacillus spores

are very dangerous because of its ability to survive in

high temperature also in the baking period which may be

germinated after baking or during storage (Legan, 1993).

Several studies on food reported that contaminated and

https://orcid.org/0000-0001-8137-3631

https://orcid.org/0000-0002-1862-7528

https://orcid.org/0000-0002-6987-4019

 Das et al. / Food Research 4 (4) (2020) 1312 - 1316

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

unhygienic products have been associated with food-

borne illness, for this reason, analyses of bakery products

are essential as these may affect the overall health of the

persons and consumers (Saranraj and Geetha, 2012).

The objective of this study was to investigate the

microbiological quality of the bakery samples collected

from the market of Dhaka city and determination of

antibiotic resistance patterns of isolated pathogens.

2. Materials and methods

2.1 Study area, sampling and sample processing

Different bakery products were collected from

markets early in the morning after production. All

samples were instantly transported to the

Microbiological Laboratory at Stamford University

Bangladesh, Dhaka. Each sample (10.0 g) was

homogenized with 90.0 mL of sterile normal saline to

prepare a stock solution. Stocks were serially diluted up

to 10-5 by adding 1 mL of samples on a series of 9 mL of

normal saline.

2.2 Microbiological analysis of each sample

A volume of 0.1 mL from each sample suspension

was spread onto nutrient agar (NA) and incubated at 37°

C for 24 hrs for enumerating total viable bacteria (TVB).

Sabouraud dextrose agar (SDA) (Oxoid Ltd.,

Basingstoke, Hampshire, England) was inoculated

followed by incubation at 25°C for 48 hrs for the

isolation of fungi (Noor et al. 2013; Rahman et al.,

2016).

2.3 Isolation pathogenic microorganisms

For the isolation of coliform bacteria (Escherichia

coli, Klebsiella spp.), 0.1 mL of each sample suspension

was spread over MacConkey (Oxoid Ltd., Basingstoke,

Hampshire, England) agar and incubated at 37°C for 24

hours.

0.1 mL of each sample suspension was spread on

mannitol salt agar (MSA) (Oxoid Ltd., Basingstoke,

Hampshire, England) for the estimation of

Staphylococcus aureus and the plates were incubated at

37°C for 24 hrs. For the estimation of starch hydrolyzing

bacteria (Bacillus spp.), 0.1 mL of each sample

suspension was spread onto starch agar (SA) (Oxoid

Ltd., Basingstoke, Hampshire, England) and incubation

at 37°C for 24 hrs.

For the enumeration of Pseudomonas spp., 0.1 mL of

each sample suspension was spread onto Pseudomonas

agar (Oxoid Ltd., Basingstoke, Hampshire, England) and

plates were incubated at 37°C for 24 hrs. All the

microorganisms were further confirmed by biochemical

methods  (Cappuccino and Sherman, 1996; Rahman et

al., 2016; Noor et al., 2013; Marjan et al., 2014; Rahman

et al., 2016; Rahman et al., 2016).

2.4 Enrichment of Salmonella spp., Shigella spp. and

Vibrio spp.

The in vitro cultivation of the species of Salmonella,

Shigella and Vibrio often appears difficult or with faulty

results (false-negative) due to their viable but non-

culturable (VBNC) attributes (Colwell, 2000; Oliver,

2005; Das et al., 2013). Consequently, enrichment was

used prior to isolating these bacteria (Rahman and Noor,

2012; Ahmed et al., 2014). For the isolation of

Salmonella spp. and Shigella spp., 1 mL of homogenized

sample suspension was transferred to selenite cystine

broth (SCB) and for Vibrio spp.1 mL of sample was

transferred to alkaline peptone water (APW) then

incubated at 37°C for 4-6 hrs. After incubation samples

were serial diluted up to 10-4and from 10-4 dilution 0.1

mL was spread onto Salmonella Shigella (SS) agar (Hi

media, India) and TCBS (Hi media, India) respectively

for Salmonella spp., Shigella spp. and Vibrio spp. (Noor

et al., 2013; Rahman et al., 2016).

2.5 Antibiotic susceptibility test

Antibiotic susceptibility traits of the pathogenic

isolates were examined by disc diffusion method where

bacterial suspension (105 cells or 0.5 OD) were prepared

from isolated pathogens. Then bacterial lawn was done

on Mueller-Hinton agar (Oxoid Ltd., Basingstoke,

Hampshire, England) plates and commercially available

laboratory-grade antibiotic discs (Ampicillin (10 µg),

Ciprofloxacin (5 µg), Streptomycin (10 µg), Ceftriaxone

(30 µg), Imipenem (30 µg), Penicillin (10 µg),

Gentamycin (10 µg), Azithromycin (15 µg), Tetracycline

(30 µg), Cefixime (5 µg), Erythromycin (15 µg) and

Trimethoprim-Sulfamethoxazole (25 µg) were

aseptically placed on the surface of agar plates at spatial

distance of 5 mm (Marjan et al., 2014).

3. Results and discussion

Different studies have observed that bakery products

are an important portion of energy intake (Agte et al.,

2002; Bartrina et al., 2004; Vanelli et al., 2005). But post

preparation contamination is a serious problem for public

health. Food safety authority’s make some food standard

the maximum permissible limits in baked products (cake,

bread and biscuits) for total plate count (TPC) is <105

CFU/g, yeast and mold is <104 CFU/g, coliform bacteria

<200 CFU/g, E. coli absent (Gilbert et al., 2000;

Hocking, 2003; NSW Food Authority, 2009).

The present study revealed that all the samples were

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found to be contaminated with heterotrophic bacteria

within the range of 104 to 107 CFU/g (Table 1). Six out

of twenty samples exceeded the microbial limit. The

proliferation of bacteria may lead to disease if bakery

items consumed by immune-compromised patient or

children which is a serious problem for the buyer. In the

case of total fungi, all the bakery items were found to

harbor microorganisms within the range of 102 to

103CFU/g which is under the microbial limit.

In the case of specific microflora the growth of

Klebsiella spp., Pseudomonas spp. are most predominate

those are confirmed by biochemical identification (table

2). Klebsiella spp. are found on all the samples within a

range of 102-105 CFU/g which is very alarming which

also sheds light on the unhygienic processing

conditioning. Pseudomonas spp. is ubiquitous organisms

which can be found everywhere, but the presence in food

was not acceptable. Here, out of ten samples, only six

contain Pseudomonas spp. E. coli was totally absent in

all the samples. Among the four samples, most of the

bread and cake were contaminated with Staphylococcus

spp. Therefore, it can be inferred that these baked

products were not handled in an appropriate manner by

the workers. Consuming Staphylococcus spp. also can

cause diseases such as nausea and vomiting, they should

use gloves and do not use outside clothing in possessing

areas. Surprisingly, Salmonella spp. also found to be

harboring in two samples, which may come from poultry

products like eggs or milk (Table 1). In the bakery, they

use egg in cake, bread and biscuits which major source

of Salmonella spp. so egg should wash before

formulation. Vibrio spp. was also present in three bakery

items and water used in bakery may the possible source

of these types of result.

Unacceptable levels of bacteria might result from

this study may be due to poor hygienic practices, from

dirty equipment and polluted environments of processing

and storage area. Compare to other sweets items harbor

less microbial count may be due to its high sugar

content. Prevalence of drug-resistance gene is growing

day by day within the human body through the ingestion

of drug-resistant bacteria by water, food and

pharmaceuticals (Ahmed et al., 2013; Hassan et al.,

2013). A different study on food water and

pharmaceuticals products revealed that they contain drug

-resistant bacteria (Noor et al., 2013; Ahmed et al., 2013;

Marjan et al., 2014). In this study similarly, all

pathogenic isolates were resistant against multiple

antibiotics tested (Table 3).  Among the five pathogens

Staphylococcus spp. and Pseudomonas spp. exhibited

more resistance against tested antibiotics which indicates

them as the MDR strain. However, other pathogenic

strain also contains resistant genes (Table 3). Abuse use

of antibiotics may be a possible reason behind the

development of drug-resistant strain which may generate

serious health threat for consumers (Marjan et al., 2014).

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Sample TVB
Total

fungus
E. coli

Klebsiella

spp.

Staphylococcus

spp.
Vibrio spp.

Salmonella

spp.

Pseudomonas

spp.

Baily road

Sweet 9.2×104 2.4×102 0 1.0×102 0 0 0 0

Biscuit 6.5×104 2.6×102 0 5.0×102 1.7×102 0 0 0

Cake  5.6×105 2.2×102 0 1.3×101 1.2×102 0 0 0

Bread 2.1×106 2.6×103 0 1.2×101 1.5×102 0 0 0

Shantinagar

Sweet 3.4×104 4.6×102 0 0 0 0 0 0

Biscuit 2.4×105 5.4×103 0 1.2×102 0 0 0 0

Cake  7.5×105 1.5×102 0 1.1×102 1.7×102 0 0 8.8×102

Bread 6.5×106 2.8×103 0 1.3×102 2.1×102 0 0 3.0×102

Mouchak

Sweet 1.9×104 5.6×103 0 0 0 0 2.0×102 0

Biscuit 2.4×105 4.8×103 0 0 0 9.5×103 3.8×103 0

Cake  5.3×105 3.2×102 0 3.6×103 2.5×102 0 0 0

Bread 4.1×106 5.2×103 0 2.3×102 1.8×102 0 0 0

Khilgoan

Sweet 1.1×103 4.6×102 0 0 0 0 0 0

Biscuit 1.9×104 1.2×103 0 0 0 0 0 0

Cake  8.6×105 3.4×102 0 3.3×102 2.1×102 0 0 8.0×102

Bread 3.9×106 4.4×103 0 5.6×101 2.2×103 0 0 4.1×102

Siddeshwari

Sweet 2.9×104 1.2×102 0 2.0×101 0 0 0 0

Biscuit 2.8×105 1.7×102 0 0 0 0 0 0

Cake  1.9×107 2.4×103 0 2.0×102 3.3×102 2.2×103 0 5.2×103

Bread 1.8×107 2.1×103 0 1.3×102 4.0×102 7.5×102 0 3.1×103

Table 1. Microbiological analysis of some bakery items

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4. Conclusion

The present study revealed the microbiological and

hygienic status of filled bakery products. Overall, the

microbiological status of filled bakery products was

slightly good while other samples were found to be

contaminated with different microbes including disease

causing bacteria with higher counts than accepted limits.

The presence of those microbes in bakery products

indicated an alarming situation for the final consumer.

So, maintenance of hygienic practice during preparation

of bakery products and use of standard procedure to

reduce the cross contamination from raw materials is

also necessary to ensure public health safety. However,

the food quality in Bangladesh needs to be monitored

regularly by BSTI and by the Ministry of Health in

Bangladesh.

Conflict of Interest

The authors declare no conflict of interest.

Acknowledgments

We thank Microbiology Laboratory, Stamford

University Bangladesh for laboratory facilities, technical

assistance and financial aid.

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CIP (5µg) 0% 100% 100% 0% 0% 100% 0% 100% 83.30% 16.70%

STE(10µg) 0% 100% 54.50% 45.50% 66.60% 33.40% 0% 100% 66.60% 33.40%

CEF (30µg) 28.60% 71.40% 81.80% 18.20% 33.30% 66.70% 0% 100% 50% 50%

IPM (30µg) 35.80% 64.20% 100% 0% 66.60% 33.40% 50% 50% 66.60% 33.40%

PEN (10µg) 57.20% 42.80% 100% 0% 100% 0% 100% 0% 83.30% 16.70%

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AZI (15µg) 28.60% 71.40% 63.60% 36.40% 0% 100% 50% 50% 33.40% 66.60%

TER (30µg) 0% 100% 36.40% 63.60% 33.40% 66.60% 50% 50% 50% 50%

CFX  (5µg) 57.20% 42.80% 72.70% 27.30% 33.40% 66.60% 100% 0% 50% 50%

ERY (15µg) 57.20% 42.80% 90.90% 9.40% 33.40% 66.60% 50% 50% 66.60% 33.40%

SXT (25µg) 0% 100% 9.40% 90.90% 33.40% 66.60% 50% 50% 16.70% 83.30%

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Sulfamethoxazole.

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