Abstract: This is the first report describing traditional technology of Dhaka cheese preparation upon surveying the study area and also some physicochemical properties of the product. Although Dhaka cheese is usually sold in unripened (fresh) condition; in the present study, an attempt was taken to investigate and compare ripening effects on physicochemical properties including proteolysis pattern of Dhaka cheese either collected from two different places of Bangladesh or prepared in the laboratory (control type Dhaka cheese). The nitrogen distribution pattern, analyzed by the Kjeldahl method showed an increase in nitrogen content and ripening index during ripening. Proteolysis pattern, carried out by Sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) displayed a higher degradation of protein in Dhaka cheese during ripening. Loss of moisture content and increase of pH and titratable acidity during ripening was also observed. However, changes of physicochemical characteristics during ripening significantly vary among the places from where Dhaka cheese collected. This result strongly indicates the microbial biodiversity and generation of short peptides in traditional Dhaka cheese during ripening.
INTRODUCTION
Several types of fermented milk products have been reported to exist throughout the world (Stanley, 1998; Tamime and Marshal, 1997). Among them, cheeses constitute one of the families of milk-derived fermented products having a number of advantages over fresh fermented products. There are many traditional cheeses from different corner of the world have been reported. The traditional food products are often considered as an indication of the cultural heritage of a nation, have persisted over centuries and they often evolved from home manufacture in an artisan and traditional way to large-scale industrial production using specific starter cultures and modern equipment (Oberman and Libudzisz, 1998). However, empirical manufacturing practices and poor sanitary conditions associated with such practices are incompatible with the tighter control enforced by Public Health Offices, a situation that will eventually hamper the global trade of such products (Tavaria et al., 2006).
Dhaka cheese is such type of traditional fermented dairy products in Bangladesh. The cheese was originated from a small village named Austagram of Bangladesh around 100 years ago. Dhaka cheese is manufactured from raw milk of indigenous cow using cattle abomasum as a source of rennet, is produced at home in small scale and is sold in unripened (fresh) condition. Special type of bamboo made basket is used for Dhaka cheese preparation. Like typical traditional fermented dairy products, for the preparation of Dhaka cheese fermentation process relies on the natural microbiota of the milk and the environment. Backslopping, a process wherein a portion of a traditionally fermented milk or sour cream from a earlier batch is used as an inoculum for the new batch, is also practiced sometimes to expedite the fermentation process.
Although Dhaka cheese has long been popular in Bangladesh, no scientific report has been found on this traditional product. Moreover, standardization of this traditional product is yet to be defined. Microbial biodiversity has recently been reported in many traditional cheeses (Ouadghiri et al., 2005; Zamfir et al., 2006; El-Baradei et al., 2007, 2008). Thus, bioactive peptides are expected to generate by the action of natural microbiota present in traditional cheese. Since Dhaka is sold in unripened condition; it would be of great importance to study the biochemical changes in this traditional fermented dairy product during ripening. Therefore, the present study was carried out to introduce traditional Dhaka cheese for the first time by describing traditional technology and also by analyzing the physiochemical characteristics including proteolysis pattern of Dhaka cheese during ripening. In addition, a control type Dhaka cheese was prepared in the laboratory and compared to traditional Dhaka cheese to observe the diversification of biochemical changes in this traditional product during ripening.
MATERIALS AND METHODS
Survey on Dhaka Cheese
The information on Dhaka cheese technology was collected by surveying
the cheese producing area in Bangladesh in the year of 2005. Interview
on several cheese makers was carried out to get the reliable data. Moreover,
to learn the know-how of Dhaka cheese preparation and also to check the
application of protocol obtained during interview, active participation
during the preparation of Dhaka cheese was followed.
Collection of Cheese Sample
Cheese samples were collected from two different places of Bangladesh
as shown in Fig. 1. The place Austagram (Fig.
1A) was selected considering origin of the cheese and named here as
DA. On the other hand, the place Manikgonj (Fig. 1B)
is known as one of the milk pocket area in Bangladesh and also a large
number of families of this area are involved with cheese making. Therefore,
Dhaka cheese sample was also collected from Manikgonj and named here as
DM. Cheese makers were requested to prepare cheese and samples were collected
soon after preparation.
| Fig. 1: | Dhaka cheese collection area, (A) Austagram, the origin of Dhaka cheese and (B) Manikgonj, one of the milk pocket area in Bangladesh. Source of image: http://www.bdshots.com/v/Bangladesh/atlas/ |
A control type Dhaka cheese, named as DC was also prepared in the laboratory using raw milk obtained from Dairy Farm of Rakuno Gakuen University, Ebetsu, Japan, commercial starter culture (Chr. Hansen`s, YO-FLEX YC-350) and rennet (Chr. Hansen`s, Rennet powder). The protocol followed by the cheese makers was applied for the manufacture of DC. Special type of bamboo made basket usually is used for Dhaka cheese preparation and was obtained from Bangladesh for the preparation of DC. In order to investigate the physicochemical changes occurred by cheese microbes, all samples were kept at 25°C (as this is the average temperature in Bangladesh) for a period of 28 days. At every 7 days interval samples were taken for further analysis.
Physicochemical Properties of Cheese
The moisture percentage of all type cheeses was obtained by drying
the samples at 100°C for 90 min using infrared moisture determination
balance (FD-600, Kett Electric Laboratory, Tokyo). The ash content was
determined by ashing the sample to constant weight at 550°C. The Total
Nitrogen (TN) and Water-Soluble Total Nitrogen (WSTN) content was determined
using the Kjeldahl method as described by Nihonyakugakkaihen (1999). The
Non-Protein-Nitrogen (NPN) and Low Molecular Peptide Nitrogen (LMP-N)
content in WSTN was also measured using the Kjeldahl method. High Molecular
Peptide Nitrogen (HMP-N) content was measured by subtracting LMP-N content
from NPN content. Water Soluble Protein Nitrogen (WSP-N) content was determined
by subtracting NPN content from WSTN content. The ripening degree was
estimated using the formula [(WSTN/TN)x100]. The total titratable acidity
(SH: Soxhlet Henkel) was determined according to Nihonyakugakkaihen (1999).
The pH of the cheese samples was measured using a pH meter (HM-5S, TOA
Electronics, Tokyo).
Analysis of Proteolysis Pattern
Sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE)
(Laemmili, 1970) was carried out to analyze the proteolysis pattern of
cheese samples during ripening. The concentration of separation gel was
15%. Each cheese sample (0.001 g) was dissolved with 2xsample buffer (0.2
mL) and then applied (10 μL) on gel. Proteins separated were stained
with Coomassie brilliant blue R250.
RESULTS AND DISCUSSION
Technology of Dhaka Cheese
A village of Bangladesh, named Austagram located 250 km North-East
of Dhaka city is the origin of Dhaka cheese. Now-a-days, Dhaka cheese
is prepared in many places of Bangladesh. The technology was diffused
gradually by migrating the cheese makers family members to the other part
of the country especially where milk production is abundant as well as
price is cheap. However, consumers still prefer to buy Dhaka cheese prepared
in Austagram.
The cheese is produced at home using the raw milk of indigenous/crossbred
cows sold by the smallholder farmers in the local market. The fourth stomach
(abomasum) of cow is collected from local slaughter house followed by
washing and sun drying with table salt and is kept until used as a source
of rennet. As shown in the Fig. 2, after filtration
using indigenous towel raw milk is coagulated with the addition of indigenous
coagulating solution. Coagulating solution is prepared by mixing previously
prepared abomasum, raw milk and water followed by 12 h incubation at room
temperature (Fig. 2). This coagulating solution is locally
known as MAWA or medicine water. Before adding, quantity of coagulating
solution is determined by checking coagulation ability of the solution
by mixing one drop of solution and around 10 drops of milk to be coagulated
on the palm. The solution is then slowly added to the milk with gentle
stirring by hand and kept undisturbed for 30 min for coagulation. After
examining the coagulation condition by entering a bamboo knife, the curd
is allowed to stand for 10-15 min.
| Fig. 2: | Schematic diagram of traditional technology for Dhaka cheese preparation |
The curd is then cut in square size using a bamboo knife and settled
for another 15-20 min. After milling using hand, the curd is kept for
further 30 min to drain out the whey. The curd is bundled using hand and
is separated from whey by curving the container. The curd is then cut
with knife and packed in to a special type of bamboo made basket by applying
pressure using hand for draining of whey. For proper shape and size turning
is made at every 4/5 h interval for a period of 16 h. After sufficient
hardening of the curd, salting process using table salt is started. Three
holes of 7.5 cm diameter each are made on the center of the upper and
lower surface of cheese at an interval of 24 h and salt is plugged into
these holes. The outer surface of cheese is also rubbed by salt at every
24 h interval for 72 h. The cheese is then ready for sale. However, cheese
surface is rubbed by salt everyday until selling. It should also be noted
that cheese is washed with water before is being appeared to the consumer
for sale.
| Table 1: | Physicochemical properties of Dhaka cheese during ripening |
| *TN: Total Nitrogen, WSTN: Water-Soluble Total Nitrogen, NPN: Non-Protein-Nitrogen, LMP-N: Low Molecular Peptide-Nitrogen, HMP-N: High Molecular Peptide Nitrogen, WSP-N: Water Soluble Protein Nitrogen | |
Changes in Chemical Composition
No significant changes of pH were observed in DM and DC type cheese.
Conversely, pH value of DA type was significantly increased after 21 days
and was again decreased after 28 days of ripening (Table
1). This increase of pH may be due to high degradation of proteins.
Presence of mold may also be the cause of such increase of pH. It is reported
that presence of Brevibacterium linens in cheese caused an increase
in pH (5.5-6.0) during ripening (Kato and Ando, 1994). In an artisanal
Mexican Fresco cheese, pH values near to 6.3 and 6.0 were also reported
(Torres-Llanez et al., 2005). However, the pH values at the beginning
and end of ripening of all cheese samples were in the range of 5.26 and
5.88 which was similar to those observed in other cow`s milk cheeses (Marcos
et al., 1981, 1983; Coulon et al., 1998; Prieto et al.,
2000). On the other hand, the titratable acidity was gradually increased
with the ripening time in all cheese
samples. The evolution of titratable acidity was less in DC and high in DA and DM cheese throughout the ripening period. However, owing to the high titratable acidity values observed, lower pH values would be expected in cheese samples. The titratable acidity-pH relationship is not constant under all conditions and might be influenced by such factors as types of organism, initial apparent acidity and buffer capacities. The apparent discrepancy in the presented data can be explained by any of these factors. During ripening, all Dhaka cheese samples lost moisture with an increase in ash content; however, rapid loss of moisture was observed after 14 days of ripening in DM and DA type and after 21 days in DC type cheese (Table 1).
Proteolysis Pattern
Nitrogen content in Dhaka cheese during ripening is also shown in
Table 1. All the nitrogen contents such as TN, WSTN,
NPN, LMP-N, HMP-N and WSTN were found to be increased with ripening period.
The TN content of DM and DC type cheese samples was little bit higher
than that of DA type. Conversely, WSTN content in DA was higher than DM
and DC. The LMP-N content of DA type was closer to DC type but higher
than that of DM type cheese sample. These data indicate the high degradation
of proteins in DA and DC type than that of DM type cheese samples.
| Fig. 3: | Changes of ripening index of Dhaka cheese during ripening |
| Fig. 4: | SDS-PAGE analysis of Dhaka cheese samples at different ripening period, (a) Control, (b) Austagram and (c) Manikgonj |
The ripening index of DA type was estimated to be higher than DC type and DC type was higher than that of DM type cheese samples (Fig. 3). This result is correspondence to the findings by SDS-PAGE analysis as shown in Fig. 4. Degradation of protein with the increase of ripening time was observed in DC and DA type Dhaka cheese (Fig. 4a, b) whereas very little degradation was observed in DM type Dhaka cheese (Fig. 4c). The data presented here indicated a higher proteolysis in DA type cheese and also a distinct difference with DM type cheese samples. Proteolysis is one of the main biochemical events that occur during cheese ripening and plays a vital role in the development of flavour (Sousa et al., 2001). Proteolysis is catalyzed by enzymes from the coagulant, milk, starter bacteria, the adventitious nonstarter microflora and in certain varieties, from the secondary microflora (Poveda et al., 2003). Since Dhaka cheese is prepared using the microflora present in raw milk and the source of milk is different between DA and DM type cheese; microbial biodiversity is expected.
CONCLUSIONS
It is generally considered that environmental conditions such as temperature, origin of the milk, processing and sanitary conditions, etc., might have a significant influence on the microbial composition of traditionally made dairy products. High degradation of proteins also is an indication of generating short peptides in DA type cheese. Therefore, existence of some bioactive peptides in Dhaka cheese may be possible. Thus, the present study is expected to encourage more research such as microbial diversity or bioactive peptides in traditional Dhaka cheese.