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In this study, the effect of different starter culture has been studied and the activity of yeast during the rice wine fermentation also being observed. The UHT milk were being mix with different kind of starter culture. Later, the yoghurt mixture were being incubated and further undergoes chilling period which results in the desired yogurt. Natural yogurt and Lactobacillus Casei Shirota had been chosen to be the best among other starter culture as the pH is drop dramatically and this proves that, the metabolite activity was high. As for rice wine fermentation, yeast was being added into the rice and being mix thoroughly. The specific gravity and pH were being observed. The pH value of the rice wine decreasing as this proves that the acidity is increasing due to the metabolite activity of yeast. The specific gravity of the yeast shows increase as this indicates that the sugar were being extracted out from the rice.
Ethanol fermentation had been found by human beings which indicates that the change that being caused by the bacterial action could results in the formation of products that were enjoyable to consume. For example, the spoilage of fruit juices can results in the formation of wine.
An early experiment carried out by Eduard Buchner in 1896 in which he grounded up a group of cell with sand until it totally being destroyed. The liquid that remain were being extracted and being added to sugar solution. He assume that fermentation could not occur since the cells are dead because there are no ‘life-force’ needed to carry out fermentation (Albasi et al., 2001).
Fermentation is the process which produces alcoholic beverages or acidic products. The fermentation which occur basically involves in the breaking down of complex organic substances into simple substances (Budslawski and Drabent, 1972). Glycolysis provide an energy towards the microorganism and causing sugar molecules to be split up and the electrons were removes from electrons to molecules (Arici et al, 2004). The electrons were being passed around to an organic molecule such as pyruvic acid resulting in the formation of a waste product such as lactic acid, ethyl alcohol and etc which is vital to utilized fermentation (Arici et al, 2004).
The production of yogurt is mainly through fermentation by lactic acid bacteria at the range of 27°C – 40°C. The mechanism by which the synergistic effect of Streptococcus spp. and Lactobacillus spp. have been well studied (Collins and Gibson, 1999). The process mainly involves in the pretreatment of milk, homogenization, heat treatment and cooling. Many study have been done towards the probiotic bacteria which can be used as the starter culture in the yogurt production which being health benefits (Shortt, 1999).
In the rice wine production, the end product which is wine is resulted from glucose fermentation. Glucose which is present in the rice will be broken down to form alcohol under favourable condition of the yeast (Arici et al., 2004). The rice wine will continue until all sugar were used up and being converted to alcohol. Based on (Arici et al, 2004), the process of producing rice wine has two steps where by saccharification process is the first step which uses fungal in order to hydrolyse the rice starch into sugar.
The next step which involves are utilizing the product of saccharification to ethanol which can be describe as below:
In this experiment, the studies aim to look the effect of different starter culture that has being used for yogurt production and to observe the rice wine fermentation in the changing of pH and specific gravity.
Materials & Methods:
- Yogurt fermentation
There were 6 different starter culture that had been used which were natural yoghurt, Pseudomonas sp., yeast, Lactobacillus casei Shirota (Yakult), Lactobacillus acidophilus (vitagen) and Lactococcus Lactis. Then, 12 mL of these starter cultures were being added into 500 mL UHT milk and being labelled separately. The pH of the milk before and after adding the starter culture were being taken. The mixture were being incubated at 40-46°C for 4-5 hours. After this incubation, the pH of the yoghurts mixture will be tested again. The yoghurts were being incubated at 5°C and the pH and also gram stained were performed after this chilling period.
Below is the flow chart which summarises the methods of yogurt fermentation:
- Rice wine fermentation
1 kg of cooked rice were being prepared and being placed in a room temperature when conducting this experiment. The rice then were mix in ground with starter culture which are yeast and gradually being added up with approximately 1L of water until all the rice were mixed thoroughly with the yeast. The pH and specific gravity of the rice were being taken every day. Below is the flow chart which summarises the methods of rice wine fermentation:
- Yogurt production
In the first session of the yoghurt production, 12ml of the starter culture which are natural yoghurt, Pseudomonas sp., yeast, Lactobacillus casei Shirota (Yakult), Lactobacillus acidophilus (vitagen) and Lactococcus Lactis being inserted into the UHT milks. Then the pH were taken and being followed by incubation at 40-46°C. The pH and visual observation were taken again and continued to under chilling stages at 5 °C. The final results and observation were taken.
The morphology of the microorganism in the yogurt mixture were tested using the gram stain methods and being visualize using the microscope. In order to specify the identity of the microorganism and the quality of the product, the pHs and the gram stain observation were being compared.
- Yoghurt production
Table 1: The result of the pH of yogurt at different stage of fermentation and the gram stain result
Starter Culture Starter pH of the UHT milk Initial pH pH after 4- 6 hours of incubation at 40-46°C pH after chilling period at 5°C Observation Gram stain observation
Natural yogurt 6.4 5.31 2.90 3.69 >5°C-sour smell, curd on top, liquid at bottom, yellowish colour
< 5°C - egg and mayonnaise like smell, white in colour and having thick consistency Two colonies (Gram positive) were found:
1) Long rods, chain
Pseudomonas sp. ~ 6 5.50 4.56 4.84 >5°C-rotten smell, liquid and curd present
<5°C-yoghurt smell, low consistency, more liquid Gram positive, long rod shape, single cell
Yeast ~ 6 5.24 4.40 4.56 >5°C-sour smell, liquid surround the curd, yellowish in colour
<5°C-sour smell, 2 phases: At the top:solid floating, bottom: liquid, yellow in colour Gram positive, cocci, chain - This are the contaminants
– no yeast are found
(Yakult) Lactobacillus casei Shirota ~ 6 5.56 3.18 3.24 >5°C-very sweet milky smell, more viscous, even consistency
<5°C - very sweet milky smell, even consistency, Gram positive with long thin rod shape colonies
(Vitagen) Lactobacillus acidophilus ~ 6 5.66 3.23 3.32 >5°C-sour yogurt smell, milky lumps, thick consistency
<5°C-yoghurt smell, more even consistency Gram positive, long rods chain
Lactococcus Lactis ~6 6.4 3.19 3.22 >5°C and <5°C observations are the same° smooth and thick liquid, cream colour, sour like smell Gram positive, cocci shape in short chain.
From table 1, all of the starter culture that was being used have a decrease in pH value from the inital pH to the incubated pH after undergo 40-46°C incubation. All of the starter culture also have an increase value after undergo 5°C chilling period. From figure 1 and table 1, the similarity and differences of morphology between all types of starter culture was being identified. When undergone gram staining, all of the starter culture showed purple colour under microscope indicates that it was gram positive bacteria instead gram negative bacteria which exhibit pink colour. Under the microscopic observation, the arrangement and shape of the bacteria was also being observed.
Rice wine fermentation
The experimentation of rice wine had been done for several days in which in the first day, the starter cultures were being mix with water and rice. The measurement of the pH and specific gravity were being taken every day until day 3.
- Rice wine fermentation
Table 2 : The result of rice wine fermentation
Day pH Specific gravity (cm) Wet mount observation Visual observation
0 (set up) 6.08 1.015 Clumping in 4 with blue stain appearance The rice has very sweet smell. The consistency is evenly distributed.
1 3.12 1.06 – The rice still have very sweet smell but even more. At the bottom of the rice has some sort of liquid look.
2 3.02 1.07 – The rice looks more liquid than the day before. The smell had a mixture of sweet and sour-like smell. The bottom part of the rice is in solid stage. The consistency is medium. The colour turning into cream colour
3 2.94 1.07 Clumping in chains There are 3 phases that can be seen which is white solid, liquid phase, and yellow slimy liquid at the bottom. The smell turning into sour like smell.
Based on table 2, the pH and specific gravity were being shown. The pH were found to be decreasing each day with the final pH was 2.94. The specific gravity shows an increasing value from 1.015 cm to 1.07 at day 2. However, the value are maintain at day 3.The appearance and morphology were being observed through wet mount techniques which use LPCB stain. The observation of yeast morphology can be seen at figure 2.
- Yogurt Production
The yogurt production mainly used microorganism in its process which these microorganism having roles in the production of the lactic acid and controlling the aroma and flavour of the yogurt. The quality of the yogurt basically depending upon the time, temperature and the types of the starter culture that were used in the process. The common starter culture that was being used are Streptococcus thermophilus and Lactobacillus bulgaricus. The need to control the optimum temperature for the activity of the microorganism is important to obtain proper yogurt taste and quality. The conditions with low temperature will cause the culture to grow slowly in order to fully acidify the milk and to achieve proper taste of yogurt. However, higher temperature will cause the starter culture to be killed and the activity of the microorganism will goes down and the proper yogurt will be failed to produce. This is the consequences that might happen when the optimum temperature is not met.
The secretion of the microorganism metabolite will contribute to the taste and flavour of the yogurt. This secretion and formation of the metabolites are basically depends on the growth rate of the microorganism. The incubation of the milk and starter culture for 40-46°C for 5 hours is the condition to assist the fermentation of the microorganism in the starter culture and producing the metabolite products. The temperature and time that is being set up in this experiments are being chosen to be the optimum condition for the fermentation to occur (Galesloot et al, 1968) and the rate of the culture metabolism would be higher (Gupta et al., 1996). If the metabolism of the culture is higher, the taste of the yogurt will be much sweeter (Galesloot et al., 1968).
When the desired pH of the acidity is reached, the yogurt was quickly being chilled at 5°C in order to suppress the fermentation process and any metabolic activity of the microorganism. This is to ensure the quality and taste of the yogurt are being maintain and no further activity of the microorganism in order to prevent the spoilage of the yogurt (De brabander et al, 1999). This is important to estimate the standard of the yogurt product.
There were 6 different starter culture that has been used in this experiment which are natural yoghurt, Pseudomonas sp., yeast, Lactobacillus casei Shirota (Yakult), Lactobacillus acidophilus (vitagen) and Lactococcus Lactis. The UHT milks that were given are at the pH of approximately 6.0 and these yogurts are being incubated in the same temperature and time.
The result for the natural yoghurt shows the decreasing pH after being incubated at 40 -46°C for 4 to 6 hours which are from pH 5.31 to pH 2.90. But there is apparent increase in the pH after the chilling period of 4°C which is to pH 3.69. The other starter culture also has the same pattern of pH which decreases after being incubated at 40-46°C and increase after the chilled period of 5°C. But for the other culture, the pH increase after undergo chilling period at 5°C does not as obvious as the natural yogurt.
The underlying reason for this condition to occur is that the production of the Carbon dioxide (CO2) due to lactose fermentation causing the pH to decline after incubation period of 4 to 6 hours at 40 – 46 °C. The growth of Lactobacillus is increasing and its predominates the whole yogurt produce more acid and causing the acids to accumulates which leads to the decrease in the pH of the yogurt for all of the starter culture that were being used in this experiment (Gomes and Malcata, 1999).
All of the starter cultures have slight increase in the pH after being chilled at 5°C and this is not as what is being expected. The chilling period of 5°C should maintain and control the acidity of the mixture by reducing the metabolism activity of the starter culture which present and thus, reducing the byproduct formation at this step (Gomes and Malcata, 1999). However, the experimental result does show an increase due to several reasons. The first reason is that, there might be contaminants or other types of the microorganism that might interrupted the activity of the Streptococcus thermophilus and Lactobacillus bulgaricus in this experiment. Hence, this interruption causing the low acidity and thus increase the pH. The other possible reason is that after the chilling period, the yogurt was not mixed causing pH gradient and the acid only concentrated at one part. But, to test the pH value using the pH meter, the yogurt was mix before testing the pH causing the acid to be distributed and resulting in the homogenous solution which was less acidic. This is why the results after chilling period for all types of starter culture are increased.
The observation that can be seen for natural yogurt is that after being incubated at 40-46°°C, the condition for which fermentation can occur is that, the yogurt have sour smell and have the appearance of milky curdles on the top and liquid at bottom and the consistency is thick and not even. Mostly of the visual observation of the yogurt with different starter culture have this curdles on the mixture and develop some sort of sour smell. This is because the acidity causing the disruption in the tertiary structure of the protein in the milk (Albasi et al, 2001). Hence, this situation produce curdles in the mixture. The smell of the mixture with different starter culture also have some sort of sour- like smell which can proved that the yogurt contain carbon dioxide.
Based on the result in this experiment, the natural yoghurt has the minimum pH compared to the other starter culture followed by yakult, lactococcus lactis and vitagen after the incubation period of 40-46°C. This is because the acidity shows that Lactobacillus predominates the yogurt mixture and produce more acid causing decreasing in the pH. The pH after the incubation at 40-46°C of Pseudomonas sp. and yeast are higher compared to the other due to the acid production is lower and thus produce less acid causing the pH to be high.
For the gram stained, the entire results shows gram stain positive in which under the microscope the image that could be seen is in purple colour. Most of the results are based on the expected. However for the yeast culture, no yeast being observed but the other types of microorganism can be seen. This is due to the presence of contaminants that might be cause during the pH measurement. At the bench, only one pH meter were provided and this will cause contamination of the pH meter resulting in the discrepancies in the result. The possible reason for this to occur is that the yeast might not being activated with the yogurt condition. This is due to the unfavourable condition for the yeast to grow. Hence, causing the other microorganism to dominate the yogurt.
The result from the experiment showed that the natural yoghurt and Lactobacillus casei shirota (Yakult) was chosen to be effective starter culture in yogurt production due to the decline in the pH which shows that the bacteria were actively producing metabolite by product during the fermentation stages occurring. The high metabolite will fasten the yogurt flavour when being added with milk and fasten the process which will prevent spoilage of food and maintain the quality of the food.
- Rice wine fermentation
For the rice wine fermentation, the pH are decreasing across the days of experimentation. This indicates that fermentation does occurs and producing acid as it by product. The metabolite activity which occurs in the fermentation are increasing, does produce more acids. The experimental results are based on what is expected.
As for the specific gravity the pattern that can be seen is where there is an increase in the specific gravity from day o which is during the set up to day 2. However, there is no increase in the specific gravity in day 3. The specific gravity of the rice wine is the lowest in day 0 indicates that there are not much glucose being released to the solution (xxx). There are increase in the specific gravity because of the sugar which are being release to the solution are increasing as time increases (Zoecklein et al, 1995). The increase in specific gravity value also indicates that the extraction of carbohydrate which are constituents in the rice to sugar are increasing due to the increase in the metabolite activity of yeast. Theoretically, the sugar should be decreasing as the time goes by as the sugar being converted into alcohol due to fermentation process (Wanapu et al, 2002). As in this experiment, in day 3 to day 4, the specific gravity is maintain and it supposed to be decreasing as the sugar being used up for alcohol production. This might happened because the rice wine has stopped the extraction of the sugar and conversion of sugar to alcohol has just begun. That is why the value does not show any changes.
This discrepancies might occur because that the rice were not evenly distributed and causing not all the sugar were being extracted and converted into alcohol. The time taken for this experiment is not enough to prove that there should be decrease value of specific gravity due to the glucose being used up.
From the experiment, the best chosen starter culture were natural yogurt and Lactobacillus Casei Shirota (Yakult) as the pH from initial pH undergo dramatic decline when fermentation process were done. The decrease in the pH value during this stage indicates the metabolic processes were being done and producing the by product which is acidic. The pH value of the rice wine fermentation also decreasing as a result of metabolic reaction occurring producing acidic by products and the increase in the specific gravity shows that the sugar are being extracted into the solution. Later, the sugar will be converted into alcohol causing the specific gravity to be no changed.
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