1
INTRODUCTION
1.1
Objective of Experiment
Chinese fermented wines are brewed directly from grains such as rice or wheat. Such
liquors contain less than 20% alcohol, due to the inhibition of ethanol fermentation at this
concentration. These wine are classified based on several factors. Among them are the liquor’s
dryness, the starter used in its production and its production method.
The three main ingredients that contribute to the unique characteristics of various Chinese
wine are the grains, the water, and the liquor starter. The water used for brewing is very important,
and should have a pH range of 6.8-7.2, with the hardness of 2-7˚DH, and a very low level of
minerals. Singapore’s tap water quality meets this requirement and will be used in this experiment.
The starter chosen for this experiment is the red starter, Hong-qu, which is a fermented product of
rice with various fungal strains of Monascus purpureus, a type of filamentous fungus. This starter
gives the wine a characteristic purple red colour and is used to give wines a unique colour and
flavour.
The water and liquor starter has been fixed in this experiment, and the aim of this
experiment was to investigate the differences in rice wine quality when different grains were used.
The colour spectra, alcohol level, soluble solid content, as well as sensory characteristics of the
wine made from the various types of rice was studied.
1.2
Key biochemical and physical transformation during the fermentation
During the fermentation, the main biochemical changes taking place are saccharification,
fermentation, and flavour development.
Saccharification
Steaming of rice results in the gelatinization of starch (nearly all amylopectin),
resulting in the swelling of starch granules and the leaching out of amylopectin. Gelatinization
increases the susceptibility of starch to enzymes. Monascus Purpureus found in the red yeast
starter produces large quantities of α-amylase, β-amylase and glucoamylase to hydrolyse starch
into fermentable sugars.
1
Fermentation
The glucose and fructose released is fermented successively by yeast. At cool
temperature of around 25˚C, saccharification and fermentation usually occur simultaneously. The
progressive saccharification of starch with slow alcohol fermentation is termed “parallel
fermentation”. This contributes to considerable ethanol production, which can be as high as 20%
(v/v) in the final fermented mash. Rice wine fermentation is conducted in a dense and mushy state
with rice grains in a ratio of 1:2 with water. This is termed semi-solid state fermentation. The dense
fermenting mash is considered beneficial in retaining large numbers of yeast cells in suspended
solid or the mash during the fermentation, which is one of the reasons why rice wine contains as
much as 20% (v/v) ethanol. For semi-solid fermentation, the mash floats to the surface easily, thus,
it is difficult to distribute the heat of fermentation evenly during the period of main fermentation.
Thus, the stirring of mash at appropriate time interval is the key to the proper control of
fermentation temperature.
Pigment
The wine becomes a unique purple-red colour because of the water soluble pigments
produced by Monascus purpureus as secondary metabolites (Juzlova et al., 1994). The mixtures of
pigments are stable from the chemical point of view. The group includes the orange pigments
called Monascorubin and Rubropunctatin, the yellow pigments called Monascin and Ankaflavin,
and the red pigments called Monascorubramine and Rubropunctamin (Erdogrul and Azirak, 2004).
Flavour
The wine has a mellow aroma and beautiful flavours, and leaves a relaxing and pleasant
aftertaste. The unique and complicated flavour was developed due to the red yeast starter and
many other natural microorganisms involved in the brewing process. The microbial metabolites
such as organic acids, amino acids, aldehydes, ketones, esters, and various alcohols formed
during fermentation contribute significantly to the flavour of the finished product.
2
MATERIALS AND METHODS
2.1
Materials
Tap water, hong qu and various types of rice were used. 8 types of rice were used: glutinous rice,
brown glutinous rice, Australian pearl rice, brown rice (Golden Peony Fragrant, Thailand), red rice,
white rice (Thailand) and black rice.
2
2.2
Fermentation Methods
0.5 kg of glutinous rice was soaked with excessive water for at least 4 hours before the rice was
steam-cooked for an hour. 100g of Hong Qu was ground to powder and added to the cooled rice
and mixed. The mixture was transferred to a clean and disinfected fermentor, and 600 mL of tap
was added and mixed. The fermentor was stoppered with air lock to ensure that it is air tight. The
mixture is left to ferment in a dark cool place for 2-3 weeks, with occasional stirring. At the end of 3
weeks, the wine was filtered and collected to measure the alcohol content, colour intensity and
soluble solid content.
2.3
Soluble solid content determination
The soluble solid content in the wines were approximated using a refractometer to measure the
specific gravity of a liquid. Results were recorded as Brix value (grams sucrose equivalent/100
grams water).
2.4
Alcohol content determination
Alcohol content was determined by the measurement of boiling point of the wine and determining
the %alcohol from the temperature temperature-concentration chart of alcohol-water mix.
2.5
UV-VIS spectra
The UV-VIS spectra was determined by the UV-VIS spectrophotometer.
2.6
Sensory characteristics
The following sensory characteristics were evaluated: Sweetness, alcohol level, bitterness, overall
liking. A 10 point category scale was used, and the sensory panel consisted of 34 panelists: 3 male
and 31 females.
3
3
RESULTS
3.1
Alcohol content
Black
Type of rice
White (Thailand)
Red
Brown
Australian pearl
White glutinous (2)
White glutinous (1)
0
10
20
30
40
50
% Alcohol (v/v)
Figure 1. Alcohol content of wines made from various types of rice.
The alcohol content of the wine fermented from the first white glutinous rice and Australian
pearl rice samples were much higher than that of the rest of the rice type, at 47.5% alcohol each.
The wine fermented from black rice had the next highest alcohol content at 21.3%, followed by
brown, white, white glutinous (sample 2) and red rice, in descending order, with alcohol content
ranging from 16.3% to 8.8%. The large difference in alcohol content between the two white
glutinous rice samples are likely to be due to experimental error, as the alcohol content from the
white glutinous rice fermentation evaluated by the sensory panel was higher for the sample 2 than
sample 1. Therefore, this brings doubt to the accuracy of the results of the alcohol determination by
boiling point, because if the difference in alcohol level was really so large between the 2 glutinous
rice sample, the sensory panel should had been able to detect it.
4
3.2
Soluble solids content
Black
Type of rice
White (Thailand)
Red
Brown
Australian pearl
White glutinous (2)
White glutinous (1)
0.0
2.0
4.0
6.0
8.0
10.0
12.0
14.0
BRIX value (g sucrose/100g H2O)
Figure 2. Soluble solids content of wines made from various types of rice.
The soluble solids content of the wines was highest in the two white glutinous rice samples,
at 11.8 and 11.0 g sucrose equivalent/100g H2O. The black rice had the next highest BRIX value,
at 10.2 g sucrose equivalent/100g H2O, followed very closely by the Australian pearl rice at 10.0 g
sucrose equivalent/100g H2O. The BRIX values of red rice, white rice and brown rice were 9.6, 9.0,
8.7 g sucrose equivalent/100g H2O respectively.
UV-VIS Spectra
UV-VIS Spectra
4.500
4.000
Absorbance
3.3
3.500
White glutinous (1)
3.000
White glutinous (2)
Australian pearl
2.500
Brown
2.000
Red
1.500
White (Thailand)
1.000
Black
0.500
0.000
400.0 450.0 500.0 550.0 600.0 650.0 700.0 750.0 800.0 850.0 900.0
Wavelength (nm)
Figure 3. UV-VIS Spectra of wines made from various types of rice.
5
The absorption maxima for the yellow, orange and red pigments produced by Monascus
Purpureus are at 410, 470 and 500nm respectively (Juzlova et al., 1994). In general, the UV-VIS
spectra of all the wines showed that there are 2 absorption peaks for the wines – one common at
492nm and the other peak occurs at 400nm for the wine made from glutinous rice and are possibly
at a wavelength smaller than 400nm for the rest of the wine. This indicates that there the yellow
and red pigments are present in the wine, and the absence of a defined peak at 470nm could be
because it is too close to 500nm and the effects were masked.
The wines from the glutinous rice fermentation showed a much greater peak maximum at
400nm and 490nm than other wines. This indicates that the red and yellow pigments are present in
much larger amounts in the glutinous rice wines than the other wines. The wines made from the
other rice, arranged in descending order of pigment content are as follows: Black > Australian
Pearl ~ White > Brown > Red.
3.4
Sensory characteristics
3.4.1
Alcohol level
Black
Types of rice
White (Thailand)
Red
Brown
Australia Pearl
White glutinous (2)
White glutinous (1)
0.0
1.0
2.0
3.0
4.0
5.0
6.0
Sensory average
Figure 4. Sensory evaluation of alcohol level in wines made from various types of rice.
No observable trend was obtained from the sensory evaluation of alcohol level in rice wines.
The sensory average for each wine was quite close to each other, although the sensory average
for alcohol level in the white glutinous rice samples were higher than the other rice wines.
6
3.4.2
Sweetness
Black
Type of rice
White (Thailand)
Red
Brown
Australia Pearl
White glutinous (2)
White glutinous (1)
0.00
0.50
1.00
1.50
2.00
2.50
3.00
3.50
Sensory average
Figure 5. Sensory evaluation of sweetness in wines made from various types of rice.
The sensory average for sweetness was highest in the second sample of white glutinous
rice. No other observable trend was observed in the sweetness level of the other types of rice
wines.
3.4.3
Bitterness
Black
Types of rice
White (Thailand)
Red
Brown
Australia Pearl
White glutinous (2)
White glutinous (1)
0.00
1.00
2.00
3.00
4.00
5.00
6.00
Sensory average
Figure 6. Sensory evaluation of bitterness in wines made from various types of rice.
The sensory average for bitterness was lowest in the second sample of white glutinous rice.
No other observable trend was observed in the sweetness level of the other types of rice wines.
7
3.4.4
Overall liking
Black
Types of rice
White (Thailand)
Red
Brown
Australia Pearl
White glutinous (2)
White glutinous (1)
0.00
1.00
2.00
3.00
4.00
5.00
Sensory average
Figure 7. Sensory evaluation of overall liking in wines made from various types of rice.
The sensory average for overall liking was highest in the second sample of white glutinous
rice, followed by the second sample of white glutinous rice. The sensory average of these two
values were much higher than the rest of the wines.
4
DISCUSSION
4.1
Alcohol Content
The alcohol content of the fermented wine is influenced by the starch content and the
starch composition in the various types of rice. Alcohol is formed as a result of the fermentation of
glucose, which in turn, is the product of starch hydrolysis. Therefore, increasing starch content
increases the substrate for fermentation to occur, resulting in more alcohol generated. Available
starch content is generally higher in milled rice than in rice with bran (Juliano, 1993). Therefore,
polished rice like glutinous rice and Australian Pearl rice had significantly larger alcohol content
than the unpolished rice like brown rice, red rice and black rice.
Another factor affecting alcohol concentration of the fermented wine is the composition of
starch in the in the rice. During the steaming of rice, gelatinization and eventually pasting occurs in
the starch granules in the rice – water is absorbed into the starch granule and the granule swell in
size until eventually it implodes, releasing most of the starch contents into the surroundings. During
8
cooling, the amylopectin and amylose molecules tend to aggregate together through the formation
of hydrogen bonds. Over a period of time, amylose retrogrades as the molecules rearrange in an
orderly fashion, resulting in a gradual increase of crystalline aggregates. Due to the highly
branched nature of amylopectin, it is less likely for its molecules to rearrange into an orderly
fashion, thus, amylopectin retrogrades very slowly. Retrogradation of amylose makes it less
susceptible for enzyme attack. Thus, saccharification is slower than in amylopectin.
In addition, amylopectin has a highly branched structure with a lot of non-reducing ends.
Therefore, enzymes like glucoamylase and β-amylase which from the non-reducing ends have a
more efficient reaction rate with amylopectin than amylose. Rice wine fermentation is a “parallel
fermentation” process in which saccharification and slow fermentation occur simultaneously. The
reduced rate of glucose production from amylose would result in slower alcohol production. Thus,
at the end of a period of 30 days, the concentration of alcohol in rice with higher amylopectin
content is higher than in rice with lower amylopectin content.
The white glutinous rice is a waxy rice, and its starch content is made up of 100%
amylopectin and no amylose. Both Australia Pearl and Black rice are short grain rice which contain
high amylopectin content. This accounts for why the alcohol content of Australia Pearl and White
glutinous rice are much larger than that of other rice. It also explains why Black rice has a higher
alcohol content compared to other unpolished rice like the Brown and Red rice. The amylopectin
and amylose composition in the various rice classes are summarised in Table 1.
Table 1
Composition of starch in different rice.
Rice Class
Waxy
Short
Medium
Long (Jasmine)
Amylose (%)
0
18-20
18-21
22-25
Amylopectin (%)
100
80-82
79-82
75-78
Source: Cooperative Research Centre for Sustainable Rice Production
The hydrometer is an instrument which measures the specific gravity of a solution. Sugar
has a higher density than water and will raise the water’s density. On the other hand, alcohol has a
lower density than water (ρethanol = 0.789 kg/m3; ρwater = 1.000 kg/m3) and hence will reduce the
density of water and will have a specific gravity reading below 1. Therefore, as fermentation
9
proceeds, the specific gravity of the solution will decrease. In the fermentation of red wine, the
alcohol content can be approximated by measuring the sucrose content at the start and calculating
the potential alcohol content that can be formed from the complete fermentation of the sucrose.
This is possible because sucrose accounts for 90% of the weight of grapes at the start of
fermentation, thus, assuming 100% efficiency fermentation, the potential alcohol content can be
calculated. Alternatively, the alcohol content can also be calculated from the difference between
the initial and final specific gravity reading by applying an appropriate correction factor.
However, in rice wine fermentation, the initial substrate is starch. As sucrose is formed from
the hydrolysis of starch, it is simultaneously fermented by yeast. Therefore, the methods above
cannot be applied here because the total fermentable sugar cannot be measured by the
hydrometer at the start of the experiment to predict a potential alcohol content; neither can the
initial and final SG method be used.
4.2
Soluble solids content
The total solid content is an expression for the combined content of all inorganic and
organic substances contained in a liquid which are present in a molecular, ionized or microgranular suspended form. Therefore, the BRIX value is more than a measure of mere sucrose
alone.
Unlike the alcohol content, the soluble solids content did not show drastic differences
between the wines produced from the different types of rice. This is because although the soluble
solids include peptides, amino acids, vitamins and organic acids, the bulk of the soluble solids
content are contributed by sugar. Since a “parallel fermentation” was occurring, the glucose
produced from the saccharification of starch was fermented to alcohol at the same time. Therefore,
there was very little build up of glucose. Thus, the soluble solids content reflect the organic acids
and peptide content built up in the wine.
4.3
UV-VIS spectra
The interpretation of UV-VIS spectra of the wines is that the type of rice did not affect the
type of pigments produced, since the wines all have the same maximum peak profile. However, the
10
type of rice did affect the production rate of the pigments, as can be observed by the different
intensity of light absorbed at the two maximas. Carbon source, nitrogen source, and pH have been
shown to influence pigment production by Monascus purpureus (Erdogrul and Azirak, 2004). The
pigments are formed as secondary metabolites (Juzlova et al., 1994). The growth and pigment
production capacity of Monascus purpureus are dependent on the nutrient composition available to
it in the mash, which differs with the type of rice used. However, it appears that red and yellow
pigment production was much higher in glutinous rice than in the other rice. The pigment contents
in the other rice were comparable. There could be some factor in glutinous rice that either
enhances the growth of Monascus purpureus or increases the production of the pigments.
4.4
Sensory characteristics
In general, the results from the sensory analysis are not reliable. This is because the
sensory panel was not trained to use the scale properly, and no reference standards were provided
at the two ends for the panelists. When analyzing the data, it must be taken into consideration that
the panelists have different subjective experience or percept to these samples, therefore, the
results generated may not be very useful. In addition, they have different methods of assigning
numbers to the percept and may not utilize the ends of the scale very well. Therefore, the results
seen in this study tend to cluster around a certain average value with large standard deviations.
While it is alright to use sensory evaluation to measure the hedonic liking of the various wines, the
instrumental analysis of the various sensory characteristics like alcohol and sweetness are better
evaluated using instrumental experimental techniques.
4.4.1
Alcohol level
The trend in the ranking of the alcohol level determined by the sensory panel was very
different from the trend determined by the boiling point determination. The sensory panel
determined the wine from the second white glutinous rice sample to have the highest alcohol
content, while in the boiling point analysis, the alcohol content of the second white glutinous rice
sample was determined to be much lower than the Australian Pearl rice and first white glutinous
rice sample. As mentioned earlier in section 3.1, the differences could be due to wrong
11
experimental techniques in the boiling point determination, or low difference threshold for alcohol
by the sensory panel which resulted in erroneous readings for the samples. However, the
conclusion is that the boiling point determination is more sensitive in the alcohol content
measurement, and subtle differences in the alcohol content in the wine is better discerned in this
method than in sensory evaluation, since the sensory panelists were not trained to evaluate the
alcohol content.
4.4.2
Sweetness
The trend observed in the sensory evaluation for sweetness in the wine is different from the
BRIX measurement, and this is expected. This is because the refractometer measures the total
soluble solids content, which includes sugars, organic acids, peptides and many other substances
in the wine. Sugar is only one component of the total soluble solids content. Therefore, it is not
unexpected that the sensory evaluation showed similar sweetness rating for the wine samples,
since sugar produced are simultaneously fermented to alcohol and unlikely to build up to a
significant difference in the wine samples. However, the sweetness rating for the second glutinous
rice sample was much higher than the rest of the wine samples.
4.4.3
Bitterness
Bitterness in the wine is likely to be caused by the bitter peptides produced during microbial
metabolism. Therefore, it is expected that grains with higher protein content, like the unpolished
grains (red rice, brown rice, black rice), will have a higher tendency to contain bitter peptides than
polished grains, because the bran has the highest protein content in rice (Juliano, 1993). In
particular, black rice contains significant amounts of the protein gluten, thus, it is expected to be
more bitter. However, the sensory evaluation showed no particular trend in the bitterness rating of
the various wines. This could be because bitterness can be affected by interactions with other
tastes – sweetness generally suppresses bitterness while ethanol enhances bitterness (Noble,
1994).
12
4.4.4
Overall liking
Besides ethanol, there are hundreds of chemical compounds which contribute to the
complex flavour of the wine. These compounds can be divided into four kinds: alcohols, esters,
organic acids, aldehydes and ketone.
The sensory evaluation of the overall liking showed that the results for white glutinous rice
were much higher than the rest of the wines. Hence, it can be concluded that white glutinous rice is
the best rice for wine making. This could be attributed to two main reasons. Firstly, the white
glutinous rice was removed of protein and oils, as the bran and hull were removed, leaving behind
starch. The microorganisms could metabolise protein and oils to bitter peptides and
aldehydes/ketones which creates undesirable flavour and odour. Thus, by removing the source of
these undesirable compounds, the overall flavour and odour of the rice is controlled. In addition,
glutinous starch contains only amylopectin, which is more available for enzymatic hydrolysis than
amylose because amylose retrogrades during cooling, making it less susceptible to enzyme
attacks. Therefore, the alcohol production rate of glutinous rice is higher. Probably, there might be
some components in the glutinous rice that enhances the production of the right flavour and odour
compounds by bacteria metabolism.
4.5
Further discussion
The mold, Monascus purpureus is critical in wine production as it supplies large quantities
of hydrolytic enzymes such as α-amylase, β-amylase, glucoamylase, protease, and lipase, which
are produced by the mold during growth and penetration of mycelium into kernels (Xu, date not
available). The enzymes produced by the molds are critical in the saccharification of the starch in
rice so that the yeasts can ferment the sugars to produce ethanol. Yeasts are only able to use
simple sugars like glucose and fructose in its fermentation of ethanol, and are not able to utilize
starch directly (Battcock and Azam-Ali, 1998). Thus, without the mold, fermentation would not
occur.
If only mold was present, the mixture produced by the saccharification of starch by the mold
enzymes would result in a solution containing many nutrients suitable for microbial growth.
However, since the mold produces substances with antimicrobial activities against many spoilage
13
microorganisms (Zheng et al., 2006), the solution will become a selective medium for the growth of
certain microorganisms. It has been shown in one study that at carbon source concentration of
50g/L, large amounts of ethanol were produced by Monascus purpureus despite aeration,
suggesting that respirofermentative metabolism was occurring (Chen and Johns, 1994). Hence,
ethanol will still be produced despite the absence of yeast.
4.6
Suggestions for improvement
Determination of soluble solids content, alcohol content, and UV-VIS Spectrum should have
been done in triplicates for more reliable results. In addition, a standardized definition of when the
boiling point starts in the alcohol determination should be used to minimize confusion between
different experimenters.
Sensory panel should be trained to use the line scale more effectively and standards
should be provided at the extreme ends of the scale for the panel to utilize the scale more
effectively. In addition, a larger sample size can be used for determination of the overall liking.
The wine can be fermented for longer period of time (about 2 months) to allow for greater
alcohol content development. In addition, after filtration, the wine can be packed into narrow
necked bottles and kept in a dark cool place for aging. During aging, the colour and flavour
development will continue, leading to a better quality wine.
The temperature of fermentation must be carefully controlled to ensure a correct mix of
microflora in the mash to develop good wine. If the temperature is too high, the fermentation will
progress too vigourously and larger quantities of undesirable flavour and aroma compounds might
be formed. In this project, the fermenting temperature fluctuates between 23 to 28˚C due to the
switching on and off of aircon in the laboratory during the day and night, the temperature
fluctuations might have led to the souring of the wine.
5
CONCLUSIONS
The production of alcohol, and pigments were the highest in the rice wine made from glutinous rice.
In addition, the glutinous rice wine was also the most well-liked among the sensory panel.
14
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Cooperative Research Centre for Sustainable Rice Production. (2003). Rice Science. Retrieved 13
Nov, 2006, http://www.ricecrc.org/reader/tg_comparison_table.htm.
Erdogrul, O., Azirak, S. (2004). Review of the studies on the Red Yeast Rice (Monascus
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Juliano, B.O. and FAO. (1993). Rice in human nutrition. Rome, Italy: Food and Agriculture
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