My Thesis: RESULTS AND DISCUSSION

CHAPTER 4

RESULTS AND DISCUSSION

 

4.1 Colony Morphology on MRS, MRS-bile and MRS-LP agar

 

MRS agar was used to enumerate total lactic acid bacteria in product A, B and C while MRS-LP and MRS-bile were used to enumerate L. acidophilus, L. casei and bifidobacteria differentially in the multiple strains product, B and C. The differential enumeration was based on the colony morphology on the media. Differential enumeration of L. acidophilus, L. casei and bifidobacteria was not required for product A because it contains only one strain of L. acidophilus. Table 4.1 shows the colony morphology on MRS, MRS-bile and MRS-LP agar.

 

Table 4.1: Colony morphology on MRS, MRS-bile and MRS-LP agar.

Media	Microorganism1	Colony
		Diameter (mm)	Color
MRS agar	total lactic acid bacteria	0.9 – 3.0	white
MRS-bile	L. acidophilus L. casei	0.9 - 1.5 0.9 - 1.3	light brown white creamy
MRS-LP	L. casei bifidobacteria	varies	varies

¹ target microorganism of the differential media

 

MRS agar was incubated in aerobic and anaerobic condition in order to obtained total aerobes and total anaerobes of lactic acid bacteria, respectively. Lactic acid bacteria in MRS agar yielded white round colonies ranging in diameter from 0.9 to 3 mm (figure 4.1). The colony appearance aerobes and anaerobes was similar for product A, B and C. In this study, product B and C, the total aerobes and total anaerobes represent the cells count of lactic acid bacteria whereas total anaerobes represent the cells count of L. acidophilus in product A.

 

Figure 4.1: Colony appearance of lactic acid bacteria in MRS agar for product B.

 

 

 

 

 

 

 

 

MRS-bile was used for differential enumeration of L. acidophilus and L. casei (Vinderola et al., 1999; Vinderola et al., 2000). Figure 4.2 shows the colony appearance of L. acidophilus and L. casei on MRS-bile agar. L. acidophilus yielded light brown colonies, ranging in diameter from 0.9 to 1.5 mm and L. casei yielded round white creamy colonies, ranging in diameter from 0.9 to 1.3 mm. Hence, both microorganisms yielded different color of colonies. The colony morphology of both microorganisms obtained from the MRS-bile agar was in accordance with Vinderola et al. (2000). He confirmed that these two distinct types of colonies were L. acidophilus and L. casei, respectively, as the colonies were examined on cell morphology (phase contrast, 1000x) and catalase test. Therefore, he reported that the differential cells count of L. acidophilus and L. casei can be assessed by their distinguishable colonies in the agar. As a result, in this study, MRS-bile agar provides a reliable bacterial count of L. acidophilus and L. casei.

 

Figure 4.2: Colony appearance of L. acidophilus and L. casei in MRS-bile agar for product B.

 

 

 

 

 

 

 

 

MRS-LP was used for differential enumeration of bifidobacteria and L. casei, based on their distinguishable colonies (Vinderola et al., 1999; Vinderola et al., 1999). In this study, product B and C yielded various sizes of colonies on MRS-LP (figure 4.3). The colonies did not give two distinct types of colonies and the identity of the colonies was obscured. The colonies were Gram positive and appeared as rod shape or Y-shape under light microscope (x1000). Although some of the colonies appeared as Y-shape (distinctive morphology of bifidobacteria) under light microscope, this did not verify the identity of these colonies because biochemical tests are required for verification. Concurrently, the various sizes of colonies yielded did not conform to Vinderola et al. (1999) and MRS-LP failed to enumerate bifidobacteria and L. casei. In this study, the differential cells count of bifidobacteria was not obtained.

 

The differential agar strongly relies on the differences in colonial morphology, which is not always a stable phenotypic trait to identify and quantify probiotic organisms in a product. The failure of MRS-LP agar as differential agar could be also due to the presence of others microorganism instead of the target microorganisms, L. casei and bifidobacteria. Product B and C consisted of S. thermophilus, L. acidophilus, L. bulgaricus and L .rhamnosus. Some of them might not be inhibited by the lithium propionate in the differential agar and thus they were present collectively with the target microorganisms in the MRS-LP. In this regard, growth requirements may vary strongly between taxa and this appears that the selective enumeration of all industrially important bifidobacteria by the use of one selective medium is very difficult to achieve (Casteele et al., 2006). As a consequence, the medium choice had to be considered on different aspects as it is very unlikely that the proposed media are suitable for the desired application.

 

Figure 4.3: Colony appearance of microorganism in MRS-LP agar for product B.

 

 

 

 

 

 

 

 

4.2 Viability of pharmaceutical probiotic products

 

In this study, the viability of pharmaceutical probiotic products was assayed by plate count method. Table 4.2 shows the viability of pharmaceutical probiotic products obtained from MRS (aerobic and anaerobic) and MRS-bile agar.

 

Table 4.2: Enumeration of total aerobes, total anaerobes, L. acidophilus and L. casei obtained from MRS agar and MRS-bile at 37°C after 72 hours.

Brand	Viable cell count (log10 CFU/g)1
	MRS		MRS-bile
	Total aerobes	Total anaerobes	L. acidophilus	L. casei
A	ND2	7.37±0.00	ND	ND
B	9.75±0.04	9.82±0.01	9.52±0.09	8.92±0.03
C	0	4.79±0.14	0	0

 

 

 

 

 

¹ Each value in the table represents the mean value ± standard deviation (SD) of three data points (triplicates).

² ND: Not determined

 

In this study, all the products (A, B and C) did not meet the claimed viability which stated on the label. Nevertheless, product A and B contained more than 10⁶ CFU/g (minimum requirement for probiotic product) whereas product C contained less than 10⁶ CFU/g.

 

Furthermore, the viability of single strain product (A) of L. acidophilus was assessed as total anaerobes because L. acidophilus grows well in the anaerobic condition (Lin et al., 2006). The viability of multiple strains probiotics products (B and C) was assessed as total aerobes and total anaerobes. The lactic acid bacteria in the products were grow in both aerobic and anaerobic condition because some of them were microaerophilic and strictly anaerobic. However, in product C, only anaerobic incubation yielded growth which meant that the probiotics were strictly anaerobic.

 

Moreover, the viability loss of the probiotic products may be caused by temperature fluctuation during stock delivery, storage and upon purchasing. Correct storage temperature of probiotic preparations is essential to maintain viable populations of freeze-dried probiotic bacteria. Besides, the rate of freeze drying and cryoprotectant also affect the viability of the products since product A, B and C are in freeze dried form. During freeze-drying, osmotic shock and membrane injury may happened. Large ice crystal formation caused damage to the cell membrane and hence reduced its viability. Besides, absence or inefficacy of cyroprotectant may lead to low viability in the products (Heckly, 1985; Egawa et al., 2005; Saarela et al., 2006; Morgan et al., 2006).

 

In addition, the prices of the pharmaceutical probiotic products do not correspond to the viability of the product. This is because product C is the most expensive but contained the lowest viability. In contrast, product A was the cheapest but rank second highest in viability whereas product B was the secondly expensive and contained the highest viability.

Although all the products did not meet the label claim for viable cells count, two of the products contained more than 10⁶ CFU/g (minimum requirement for probiotic product). Therefore, product A and B retain the criterion of 10⁶ CFU/g and may confer health effects upon ingestion.

 

Eventually, the prophylactic and therapeutic effects of probiotics will be effective only if the probiotics remain as high number of viable cells in the gastrointestinal tract after consumption. The minimum viable cells that is required is 10⁶ colony forming unit per gram (CFU/g) (Dave et al., 1997; Kailasapathy et al., 2000). Product A and B contained more than 10⁶ CFU/g and hence, these probiotics may exert its effect if they were able to tolerate to low pH. Subsequently, this gave the rationale of acid tolerance test.

4.3 Direct Count Method: Dye Exclusion Test

 

The dye exclusion test was tested in order to obtain direct viable counts of bacteria in the products. From the result, clumping of cells were observed under the microscope (x 400) and hence, the cells count could not be determined. Although the sample was centrifuged prior the test, the similar results were obtained. Thus, this method did not provide a reliable way of direct viable cells count.

 

The factors that affect the efficacy of this method could be caused by the presence of adjuncts (cryoprotectants) in the pharmaceutical probiotics which causes the cells clumped together. Moreover, the adjuncts may be too dense to be separated through centrifugation. Even after the sample centrifuged, the adjunct could be retained in the bacteria pellet. Since this method gave a negative result and was not reliable, this method was dropped from the study.

Therefore, the viable cells count of the probiotics was assessed by plate count method. The CFU per gram provide the viable cells count of probiotics throughout this study.

4.4 Acid Tolerance of Pharmaceutical Probiotics

 

In order to evaluate the acid tolerance of pharmaceutical probiotics, product A, B and C were tested in acidified-PBS in pH 2, 3, 4 and 6. Acidified-PBS was used instead of acidified-MRS broth because MRS broth may provide protection to bacteria by providing energy and metabolic precursors, thus potentially enhancing bacterial survival (Corcoran et al., 2005). Products A, B and C were tested for 0, 1.5 and 3 hours to simulate pH of stomach by taking into account the general gastric transit time. Table 4.3 shows the viable cells count of product A for acid tolerance test. From the study, there was significant decreased in viability at pH 2 and 3 while no significant decreased in viability at pH 4 and 6 in product A. The tolerance to pH 4 and pH 6 for probiotics product A were better than pH 2 and pH 3. At pH 2, viability of product A decreased approximately 3-fold from 6.77 log₁₀ CFU/g to 6.28 log₁₀ CFU/g after 3 hours exposure. At pH 3, viability of product A decreased approximately 2-fold from 7.07 log₁₀ CFU/g to 6.80 log₁₀ CFU/g after 1.5 hours exposure and the viability did not further decrease for another 1.5 hours. Although there were decreased in viability at pH 2, the viable cells count still maintained above 10⁶ CFU/g (minimum requirement for probiotic products) after 3 hours exposure. Thus, product A which contains probiotic strain L. acidophilus was tolerant to the simulated gastric pH.

 

Table 4.3: Enumeration of L. acidophilus in product A following exposure to pH 2, 3, 4 and 6 after 0, 1.5 and 3 hours incubation. The values are the mean of three determinations.
¹

pH	Viable cells count (log10CFU/g)2
	0hr	1.5hr	3.0hr
2	6.77±0.06a	6.99±0.07b	6.28±0.02c
3	7.07±0.16ab	6.80±0.12 abc	6.76±0.02bc
4	7.08±0.07abc	7.21±0.11abc	7.17±0.17abc
6	7.38±0.13abc	7.37±0.12abc	7.32±0.06abc

 

 

 

 

 

 

 

 

 

 

¹ Each value in the table represents the mean value ± standard deviation (SD) of three data points (triplicates/products). Means in row with common letters do not differ (P> 0.05).

² Enumeration was done using MRS agar.

 

The method for acid tolerance test was similar for all the products except in product B and C, the enumeration of probiotics included differential counts of L. acidophilus and L. casei. Table 4.4 shows the viable counts in product B following exposure to pH 2 for 0, 1.5 and 3 hours. In product B, there was significant decreased in total viability at pH 2 after 3 hours. The viable cells count of total aerobes decreased approximately 160-fold from 9.00 log₁₀ CFU/g to 6.80 log₁₀ CFU/g after 3 hours exposure. The viable cells count of total anaerobes decreased 130-fold from 9.47 log₁₀ CFU/g to 7.37 log₁₀ CFU/g after 1.5 hours exposure and the viability did not further decrease for another 1.5 hours. The viable cells count of L. acidophilus in product B decreased approximately 180-fold from 9.13 log₁₀ CFU/g to 6.88 log₁₀ CFU/g after 3 hours exposure. The viable cells count of L. casei decreased 120-fold from 8.90 log₁₀ CFU/g to 6.81 log₁₀ CFU/g after 1.5 hours exposure and the viability was maintained for another 1.5 hours exposure. Although there were decreased in total viability at pH 2, the viability still maintained above 10⁶ CFU/g (minimum requirement for probiotic products) after 3 hours exposure. Thus, pharmaceutical probiotics of product B was tolerant to the simulated gastric pH.

 

Table 4.4: Enumeration of viable count in product B following exposure to pH 2 for 0, 1.5 and 3 hours incubation.

	Viable cell count (log10CFU/g) 1
	0hr	1.5hr	3.0hr
total aerobes2	9.00±0.11a	6.40±0.07bc	6.80±0.34bc
total anaerobes2	9.47±0.04a	7.37±0.03bc	7.34±0.05bc
L. acidophilus3	9.13±0.09a	6.58±0.36bc	6.88±0.16bc
L. casei3	8.90±0.23a	6.81±0.22bc	6.81±0.22bc

¹ Each value in the table represents the mean value ± standard deviation (SD) of three data points (triplicates). Means in the same row with common letters do not differ (P> 0.05).

² Enumeration was done using MRS agar

³ Enumeration was done using MRS-bile agar

 

 

Table 4.5 shows the viable cells count of total aerobes, total anaerobes, L. acidophilus and L. casei in product B following exposure to pH 3 for 0, 1.5 and 3 hours incubation. There was significant decreased in viability at pH 3 for 0, 1.5 and 3 hours for total aerobes, total anaerobes and L. casei. The viable counts of total aerobes decreased 3-fold from 9.50 log₁₀ CFU/g to 9.08 log₁₀ CFU/g after 1.5 hours exposure and the viability was maintained for another 1.5 hours exposure. The viable counts of total anaerobes decreased 2-fold from 9.44 log₁₀ CFU/g to 9.18 log₁₀ CFU/g after 3 hours exposure. Likewise, the viable cells count of L. casei decreased 3-fold from 9.22 log₁₀ CFU/g to 8.80 log₁₀ CFU/g after 1.5 hours exposure and the viability was maintained for another 1.5 hours exposure. In contrast, L. acidophilus showed no significant decrease at pH 3 and the viability was well maintained for 3 hours exposure.

Table 4.5: Enumeration of viable count in product B following exposure to pH 3 for 0, 1.5 and 3 hours incubation.

	Viable cell count (log10CFU/g)1
	0hr	1.5hr	3.0hr
total aerobes2	9.50±0.08a	9.08±0.17bc	9.01±0.07bc
total anaerobes2	9.44±0.02a	9.27±0.12abc	9.18±0.04bc
L. acidophilus3	8.76±0.39abc	8.86±0.09abc	8.86±0.09abc
L. casei3	9.22±0.09a	8.80±0.04bc	8.80±0.04bc

¹ Each value in the table represents the mean value ± standard deviation (SD) of three data points (triplicates). Means in the same row with common letters do not differ (P> 0.05).

² Enumeration was done using MRS agar

³ Enumeration was done using MRS-bile agar

 

 

Table 4.6 shows the viable cells count of total aerobes, total anaerobes, L. acidophilus and L. casei in product B following exposure to pH 4 for 0, 1.5 and 3 hours incubation. In product B, there was significant decreased in viability at pH 4 for 0, 1.5 and 3 hours for total anaerobes only whereas total aerobes, L. acidophilus and L. casei showed no significant decrease. The viable cells count of total anaerobes decreased approximately 1.5-fold from 9.43 log₁₀ CFU/g to 9.27 log₁₀ CFU/g after 1.5 hours exposure and the viability was maintained for another 1.5 hours exposure. From the result, the viable cells count of aerobes, L. acidophilus and L. casei were well maintained at pH 4 for 3 hours exposure. Moreover, pH 4 did not cause detrimental effect on L. acidophilus and L. casei of product B.

 

Table 4.6: Enumeration of viable count in product B following exposure to pH 4 for 0, 1.5 and 3 hours incubation.

	Viable cell count (log10CFU/g)1
	0hr	1.5hr	3.0hr
total aerobes2	9.67±0.17ab	9.46±0.02abc	9.24±0.02ac
total anaerobes2	9.43±0.05a	9.27±0.08bc	9.16±0.03bc
L. acidophilus3	9.42±0.02abc	9.00±0.32abc	8.87±0.50abc
L. casei3	8.62±0.15abc	8.30±0.30abc	8.70±0.00abc

¹ Each value in the table represents the mean value ± standard deviation (SD) of three data points (triplicates). Means in the same row with common letters do not differ (P> 0.05).

² Enumeration was done using MRS agar

³ Enumeration was done using MRS-bile agar

 

 

Table 4.7 shows the viable cells count of total aerobes, total anaerobes, L. acidophilus and L. casei in product B following exposure to pH 6 for 0, 1.5 and 3 hours incubation. The probiotics of product B showed no significant decreased in viability to pH 6 for 3 hours exposure. The viable cells count of aerobes, anaerobes, L. acidophilus and L. casei were well maintained at pH 6 for 3 hours exposure. Moreover, pH 6 did not cause detrimental effect on pharmaceutical probiotics of product B.

 

Consequently, both pH 4 and pH 6 did not cause detrimental effect on aerobes, L. acidophilus and L. casei of product B after 3 hours of acidified-PBS exposure. This result was in agreement with Liong et al. (2005) where they concluded that L. acidophilus and L. casei strains were able to survive under acidic environment. Nevertheless, total anaerobes of product B were maintained only at pH 6. Although the viability of product B was decreased to variable extent, its viability was still maintained above 10⁶ CFU/g (minimum requirement for probiotic products). Therefore, pharmaceutical probiotics of product B survived the pH 2, 3, 4 and 6.

 

Table 4.7: Enumeration of viable count in product B following exposure to pH 6 for 0, 1.5 and 3 hours incubation.

	Viable cell count (log10CFU/g)1
	0hr	1.5hr	3.0hr
Total aerobes2	9.75±0.04abc	9.64±0.05abc	9.55±0.19abc
Total anaerobes2	9.82±0.01abc	9.66±0.06abc	9.57±0.21abc
L. acidophilus3	9.85±0.02abc	9.77±0.04abc	9.81±0.06abc
L. casei3	9.33±0.54abc	9.66±0.47abc	9.65±0.50abc

 

 

 

 

 

 

 

 

 

 

 

¹ Each value in the table represents the mean value ± standard deviation (SD) of three data points (triplicates). Means in the same row with common letters do not differ (P> 0.05).

² Enumeration was done using MRS agar

³ Enumeration was done using MRS-bile agar

 

 

 

On the other hand, product C was tested for acid tolerance although the initially viability was lower than 10⁶ CFU/g. Table 4.8 shows the viable cells count in product C for acid tolerance test. The acid tolerance test showed that the pharmaceutical probiotics of product C did not survive the pH 2, 3 and 4. However, in pH 6 the viability was maintained and shown no significant decreased after 3 hours exposure. From the results, probiotics of products C did not tolerate to low pH.

Table 4.8: Enumeration of viable cells count in product C following exposure to pH 2, 3, 4 and 6 after 0, 1.5 and 3 hours incubation. The values are the mean of three determinations.
¹

pH	Viable cells count (log10CFU/g)2
	0hr	1.5hr	3.0hr
2	0	0	0
3	0	0	0
4	0	0	0
6	4.79±0.01abc	4.71±0.01abc	4.69±0.01abc

 

 

 

 

 

 

 

 

 

 

¹ Each value in the table represents the mean value ± standard deviation (SD) of three data points (triplicates/products). Means in row with common letters do not differ (P> 0.05).

² Enumeration was done using MRS agar under anaerobic condition.

 

 

 

 

 

 

 

 

 

 

 

 

 

This study compared the effects of different pH on the viability of pharmaceutical probiotics in three different brands during 3 hours of acidified-PBS exposure simulating the gastric pH. In this regard, probiotics of product C did not survive at pH 2 (the lowest pH in this study) compared to product A and B. As shown in graph 4.1, product B had the highest tolerance to pH 2 compare to product A. From the result, the varied degree of acid tolerance among the probiotic products could be influenced by the different strains incorporated in the particular products (Cotter et al., 2003). Product B could be consisted of several good strains of probiotics that exhibit better survivability to low pH whereas in product C, the probiotic strains were poor and thus did not survived in low pH. Moreover, the acid tolerance of product B was due to the overall effects exhibited by all the microorganisms present inside. Hence, acid tolerance of product A was weaker than product B because its survivability in low pH was exhibited by the single strain of L .acidophilus.

 

In addition, the prices of the products do not correspond to the acid tolerance of its probiotics. Since product C is the most expensive but contained the lowest acid tolerance. In contrast, product B was the secondly expensive and contained the highest acid tolerance whereas product A was the cheapest but rank second highest in acid tolerance.

 

Low pH condition of stomach causes loss of viability and cell damage. Cell damage is envisioned as the derangement of membrane structures as well as solute leakage from the cell.
It was reported that L. casei leak magnesium only at pH less than 3. This phenomenon was comparable with the present study as the viability of L. casei for product B was affected at less than 3 (pH 2 and 3). In addition, the susceptibility to
membrane damage caused by acidification is varied between species and correlate with the degree of acid tolerance (Hutkins et al., 1993).

 

There are various underlying factors and mechanisms that could affect the acid tolerance of probiotics. The survival of probiotics is high when there are availability of nutrients, low or absence of inhibiting compounds and maintenance of hydrogen ion concentration above the level that a specific strain can tolerate. Low pH is growth limiting for probiotics and loss of cell viability may also occur in cells that are held at low pH. The acid tolerance of lactobacilli or probiotic is attributed to the presence of a constant gradient between extracellular and cytoplasmic pH (or intracellular pH). When the internal pH reaches a threshold value, cellular functions are inhibited and the cells die (Corcoran et al., 2005). Moreover, the optimum growth of bacteria occurs within a specific pH range, depending upon the species of bacterium (Nannen et al., 1991). This explains the reason why the pharmaceutical probiotics in this study have varying degree of acid tolerance.

 

In general, bacteria are equipped with a number of mechanisms that confer acid tolerance. These include proton translocation, arginine deaminase (ADI) pathway, amino acid decarboxylation-antiporter reactions, and the citrate transport system. These mechanisms will activated when bacteria are confronted with a change in the pH. Nevertheless, among these mechanisms, the one being significant to lactic acid bacteria or probiotics is the proton translocation. This is one of the important mechanism of which probiotics used for its pH homeostasis. The F₀F₁ ATPase functions to maintain a favorable intracellular pH and protect cells during exposure to acidic environment by translocating protons to the environment at the expense of ATP. F₀F₁-ATPase is induced at low pH, and regulation appears to occur at the transcriptional level (Corcoran et al., 2005). The membrane bound proton-translocating ATPase (H⁺-ATPase) extrudes protons out of the cell via ATP hydrolysis. The expulsion and movement of protons from the cytoplasm into the medium of the cells (more acidic) is against the concentration gradient and hence energy is needed. The main function of H⁺-ATPase in glycolytic, nonrespiring bacteria (lactic acid bacteria) is the maintenance of ∆pH (pH gradient) and functions as a proton pump (Hutkins et al., 1993; Matsumoto et al., 2004).

 

Furthermore, the undissociated form of an organic acid is assumed to be the most toxic form for microorganisms compare to inorganic acid, HCl. Organic acids inhibit microorganisms by entering the cell in the undissociated form and then dissociating within the cell which lead to acidification of the cytoplasm and collapse of the proton motive force, resulting in inhibition of nutrient transport. Therefore, the uses of acid in acid tolerance test simulating the stomach condition is an important consideration (Hutkins et al., 1993; Matsumoto et al., 2004; Cotter et al., 2003).

 

When probiotics are to be allowed to grow in acidic condition, it will perform the acid tolerance mechanism to maintain the cytoplasmic pH relatively constant over a wide range of environmental pH. And this will restore its pH homeostasis. But, when there is a pH shift during the stationary phase of probiotics, the expression of proteins will be modified. This inducible survival mechanism is known as acid tolerance response. Hence, it is advisable to grow cells for probiotics purposes at lower pH in order to improve the resistance of the cells to acid stress during the acidic stomach passage (Hutkins et al., 1993; Matsumoto et al., 2004; Collado et al., 2006).

 

 

 

The results from present study indicate that the survival of pharmaceutical probiotics in stomach acidity was varied among the products. The sum of these effects were influence by the strain of probiotics, the pretreatment of probiotics before undergo processing and its mechanisms of the acid tolerance.

 

Besides, there are other factors that affect the survival of probiotics in human stomach. These factors are due to various chemical components present in human stomach. These include the gastric enzyme such as pepsin as well as digested foods or nutrient. The foods matrix may protect the probiotics from the digestive enzyme as well as the nature of food will affects the transit time through the stomach. Basically, food remains in the stomach between 2 and 4 hour however, liquids empty from the stomach faster than solids, and only take about 20 minutes to pass through the stomach (Ouwehand et al., 2002; Holzapfel et al., 1998; Huang et al., 2004).

 

Although pH could be used as a suitable parameter for selection of probiotic strains, the pharmaceutical probiotics are consumed in capsule form. As such, the presence of capsule may improve the viability of microorganisms during gastric transit. This suggest that low-acid tolerant strains need not be excluded from probiotic application, providing they can be delivered to the intestine in high numbers, and preferably as part of a buffered food or encapsulated delivery system (Huang et al., 2001).

4.5 Morphology Identification

    

The colonies yielded in MRS and MRS-bile agar was tested for Gram staining. From the results, all the microorganisms from MRS agar were Gram positive. Rod and cocci shape were found in product C because product C was claimed to contained lactobacilli and S. thermophilus (table 3.1) In contrast, rod shape was found in product A and B because both of them were claimed to contained lactobacilli (table 3.1). Furthermore, all the microorganisms from MRS-bile agar were Gram positive rod shape as this media was used to enumerate L. acidophilus and L. casei differentially.

My Thesis: CONCLUSION AND FUTURE PERSPECTIVES

CHAPTER 5

CONCLUSION AND FUTURE PERSPECTIVES

 

Although several probiotic strains have been identified with health benefits, for a strain to be beneficial, it must fulfill certain criteria to be considered a valuable food to exert a positive influence. The present study was undertaken to evaluate the viability of pharmaceutical probiotic products upon reaching consumer and the survival of this probiotics to low pH. In fact, low pH is the initial critical condition encountered by the probiotics upon ingestion. Throughout the study, all the products did not meet the claimed viability. Nevertheless, two of the product (A and B) meet the minimum requirement for probiotic products (CFU/g) whereas product C did not. Product B has the highest viability and highest acid tolerance whereas product C has the lowest viability and lowest acid tolerance. The loss of viability of probiotics at low pH corresponds to its acid tolerance and hence, low pH affects the viability to a certain extent. The viability of probiotic was affected by acidic condition (pH 2, 3 or 4) and unaffected by slightly acidic condition (pH 6).

 

The products tested in this study are similarly manufactured as freeze-dried forms but the level of acid tolerance was not similar among these products. This indicates that the acid tolerance of probiotics in the product was varied in their ability to tolerate low pH. Product C confer poor tolerance to low pH suggested that the overall of probiotic strains found in the products were not tolerant to low pH whereas product B confer good tolerance to low pH suggested that the overall of probiotic strains found in the products were tolerant to low pH.

 

The acid tolerance exhibited by probiotics of product A was not good as compared to product B. Product B was better tolerant to low pH compared with product, which indicate that the application of a consortium of strains were more likely to possess better acid tolerance. Moreover, given that the human stomach is a hostile environment, it appears unlikely that a single probiotic bacterial strain will be capable of influencing the microbial ecology of the host and of beneficially affecting the host. Hence, it is essential that these probiotic strains not be developed as individual entities but rather as the active ingredients of the food products that are ultimately intended for human consumption.

 

The apparent poor results of this study indicate that several pharmaceutical probiotics could successfully transit the human stomach and may be capable of reaching the intestinal environment and functioning effectively in the intestine. However, criterion of acid tolerance for this in vitro selection could not reveal the capability of probiotic strains individually. Likewise, this is also the limitation of this study whereby selective enumeration of probiotics was used for L. acidophilus and L. casei. The bifidobacteria did not enumerated differentially from the total anaerobes because lack of reliable a selective media. Thus, the tolerable pH of bifidobacteria could not be determined and the acid tolerance of pharmaceutical probiotics is not fully understood. Moreover, the limitation of fund also restricts the uses of biochemical test such as API kit to further confirm the identity of species.

 

In conclusion this in vitro test provides an initial understanding on the acid tolerance of pharmaceutical probiotics. From this set point, in vitro acid tolerance test allow the continuation on in vivo test to fully investigate the tolerance of probiotics in human stomach. Concurrently, acid tolerance is recommended to proceed for the bile tolerance test as the second criteria.

Indian Cooking-Superb Recipe

Superb recipe from Indian Cooking Recipe Books. I like it!

Stir-Fried Zucchini

This is absolutely a healthy and low calories dish with oriental touch. Cook zucchini with stir fry method is indeed minimise the nutrients loss.

 

Recipe:

One medium size zucchini (sliced)

0.5 cup of hot water

3 cloves of grated garlic

3 tbsp of soy sauce

2 tbsp of vegetables oil

A pinch of salt (to your liking)

 

Heat the pan and pour 2 tbsp of oil. Saute the garlic for a second (until its release it's fragrant, but not burnt). Put all the zucchini's slices and stir it. Stir every 30 seconds to ensure it didn't burnt. Cook about 5 minutes or until the zucchini soften. Pour all the soy sauce and stir constantly. After a minute, pour the hot water and season with salt. Serve.

 

Why Zucchini?

Everyone knows that vegetable benefit human being especially green vegetables. There's no doubt about it. Here, zucchini provide you vitamin A, thiamine, riboflavin, niacin, vitamin B6, vitamin B12, biotin, vitamin C, vitamin D, vitamin E, folate, vitamin K, pantothenic acid, calcium, copper, iron, magnesium, potassium and zinc. Zucchini contain significant amount of vitamin C which mean it help to prevent diseases, like scurvy, bruising etc, caused by the deficiency of Vitamin C. In addition, a combination of Vitamin C and lutein in zucchini confer the antioxidant and anti-inflammatory properties that also good for eyes. Zucchinis have high water content (over 95%) and naturally pre-packaged with fiber that they make perfect food for people on diet ( or constipation..hahhaahah~)

So, why do we still need to waste money to opt for other expensive food to get this all these good nutrients for our body instead we can cook healthy dish easily at home.

 

 

 

 

 

My Thesis: MATERIALS AND METHODS

CHAPTER 3

MATERIALS AND METHODS

 

3.1 Pharmaceutical Probiotic Products

 

Three brands of commercial pharmaceutical probiotic products of lyophilized form enclosed in capsules were purchased for this study. Description and composition labeled on these products are listed in Table 3.1. These products were purchased from the pharmacies around Klang valley before their expiration date. The products were stored at refrigerator 4°C until use.

 

Products	Form	Probiotics	Viability (CFU/capsule)
A	capsule	L .acidophilus	4x109
B	capsule	L. acidophilus, L. bulgaricus ,L. casei L .rhamnosus, B. bifidum, B. infantis, B. longum.	5x109
C	capsule	L. acidophilus, L. bulgaricus, L. casei, S. thermophilus, B. longum.	1x1010

 

Table 3.1: The pharmaceutical probiotic products used in this study.

 

 

3.2 Culture Media and Reagents

 

MRS agar (de Man-Rogosa Sharpe agar; Oxoid Ltd., Hamphire, England) was purchased from Oxoid ltd. Bile bovine, trypan blue and sodium propionate were purchased from Sigma Chemical Co. (Germany). Lithium chloride was purchased from AJAX Finechem Ltd. The phosphate buffer saline (PBS) was used as the buffer solution for serial dilution and was formulated from 0.144 g/L of KH₂PO₄, 9 g/L of NaCl and 0.795 g/L of Na₂HPO₄ (Fisher Scientific Inc.(US). Hydrochloric acid was purchased from Fisher Scientific Inc.(US).

 

 

3.3 Media preparation

 

MRS agar

MRS agar was prepared according to manufacturer instruction. An amount of 31g MRS (solid-powder) was added to 500mL distilled water in a Schott bottle and mixed thoroughly. Then, the mixture was autoclaved at 121°C for 15 minutes. MRS agar contains polysorbate, acetate, magnesium and manganese, which are known to act as special growth factors for lactobacilli, as well as a rich nutrient base. Then, MRS agar (liquid) was poured into sterilized Petri dishes. About 10 to15 mL of MRS agar (liquid) were sufficient for a single Petri dish. The agar dishes were allowed to solidify and stacked invert. MRS-agar (solid) plates were use directly or packed in plastic beg and stored in refrigerator.

 

 

MRS-bile agar

The formulation of MRS-bile was prepared according to Vinderola et al. (1999) where 0.75g (0.15%) of bile and 31g MRS (solid-powder) were added to 500mL distilled water in a Schott bottle. The mixture was mixed thoroughly and autoclaved at 121°C for 15 minutes. Next, sterilized MRS-bile agar (liquid) was poured into sterilized Petri dishes. About 10 to15 mL of MRS-bile (liquid) was sufficient for a single Petri dish. The MRS-bile agar (solid) plate was allowed to solidify and stacked in invert position (Vinderola et al., 1999; Vinderola et al., 2000).

 

MRS-LP agar

The formulation of MRS-bile was prepared according to Vinderola et al. (1999) where 0.2% of lithium chloride (solid-powder) and 0.3% of sodium propionate (solid-powder) were added to the MRS basal medium. 1g of lithium chloride and 1.5g of sodium propionate were added to 31g MRS (solid-powder) were added to 500mL distilled water in a Schott bottle (Vinderola et al., 1999; Vinderola et al., 2000). The mixture was mixed thoroughly and autoclaved at 121°C for 15 minutes. Next, sterilized MRS-LP agar (liquid) was poured into sterilized Petri dishes. About 10 to15 mL of MRS-LP (liquid) was sufficient for a single Petri dish. The MRS-LP agar plate was allowed to solidify and stacked in invert position.

 

 

3.4 Sterilization of Glassware and Aseptic Environment

 

The materials and apparatus were sterilized before use. Eppendorf tubes were placed in a beaker and covered with aluminum foil for autoclave. Universal bottles, micropipette tips, PBS and tubes rack were autoclaved at 121°C for 15 minutes.

 

The plating method was conducted in laminar flow. For aseptic environment, laminar flow was sterilized ultra violet (UV) light for 15 minutes. Subsequently, vortex, Bunsen burner, micropipette and all the glass ware were wiped with methylated spirit before use.

 

 

3.5 Viability of Pharmaceutical Probiotic Products

 

3.5.1 Plate Count Method

 

For total viable counts of each product, the probiotics powder was taken out from the capsule. Instantly, 0.1g of the homogenized powder was diluted with 9ml of sterilized PBS and vortex (Boeco, Germany) thoroughly. Subsequently, 0.1 ml of the aliquot was transferred to 0.9 ml of PBS in Eppendorf tube for serial dilution. The dilution was made for 10⁷-fold. Then, 0.1ml of the appropriate dilution was spread plated on the MRS agar, MRS-bile and MRS-LP, respectively (appendix 1). For each media, triplicate results were obtained.

Plates of MRS agar were incubated at 37°C under anaerobic and aerobic condition for 48 hours. Plates of MRS-bile agar were incubated at 37°C under aerobic for 48 hours. Plates of MRS-LP agar were incubated at 37°C under anaerobic condition for 48 hours. The anaerobic condition was achieved by use of an anaerobic jar (Merck Ltd., Darmstadt, Germany) and Anaerocult® A gas pack (Merck Ltd., Darmstadt, Germany). Anaerocult® A gas pack (Kieselguhr, iron powder, citron acid and sodium carbonate) bind to oxygen efficiently to create anaerobic condition.

 

 

3.5.2 Direct Count Method: Dye Exclusion Test

 

This staining method is based on "dye exclusion" cells where intact membranes do not take up the dye and are considered viable. Trypan blue staining with hemocytometer provides a method for distinguishing between viable and nonviable cells. The chemicals used were Trypan blue (0.4%) and PBS.

 

An amount of 1g of probiotics powder was added to 9ml of PBS in a universal bottle. Then, 0.1ml from the diluted sample was added to 0.9ml of PBS in Eppendorf tube for serial dilution. The serial dilution was performed until 10⁷-fold of dilution was obtained. For each dilution, 0.5ml of trypan blue and 0.3ml of PBS was added to 0.2ml of diluted sample. The mixture was allowed to stand for 5 to 15 minutes. One drop of the mixture was transferred with Pasteur pipette to the hemocytometer. The viable cells which are unstained and nonviable cells which are stained were observed under microscope (Nikon, Japan). The observation was recorded.

 

The similar test was repeated by centrifuged the probiotics with PBS suspension prior to serial dilution. The PBS was added to probiotics powder and were centrifuged at 13 000 rpm for 1 minutes. The sample was washed with PBS for three times. Then, the sample was serially diluted and stained with trypan blue. One drop of the sample was transferred to hemocytometer. The observation was recorded.

 

Calculation:

Cell viability (%) = total viable cells (unstained) X 100

total cells(stained and unstained)

 

 

 

3.6 Preparation of Low pH PBS

 

The pH of PBS was adjusted to pH 2, 3, 4 and 6 with 0.1 N acid hydrochloric (HCl) using Model 3501 pH meter (Lin et al., 2006; Shuhaimi et al., 1999). The acidified-PBS was autoclaved as 121°C for 15 minutes. The solution was allowed to cool to room temperature before the assay.

 

 

 

3.7 Acid Tolerance Test Simulated low pH

 

One gram homogenized probiotics powder was mixed with 9 ml of acidified-PBS of pH 2.0, 3, 4 and 6 respectively (Lin et al., 2006; Shuhaimi et al., 1999; Corcoran et al., 2005). For each pH of acidified-PBS, the mixture was incubated at 37°C for 0 hour, 1.5 hours and 3.0 hours. After incubation, viable bacterial counts were determined by plating serial dilutions (with PBS, pH 7.2) on MRS agar, MRS-bile and MRS-LP. The assay was performed in triplicate.

 

 

3.8 Morphology Identification

 

    The colonies were selected randomly for Gram staining. All colonies were confirmed as Gram positive with rod shape bacteria under microscope (1000X).

 

 

3.9 Statistical Analysis

 

The data were calculated with mean values and standard deviations in triplicate trials. Data generated from the experiments were analyzed for significance by the ANOVA-one way and Tukey's for multiple comparisons. Differences were considered significant at P<0.05.