The Effect of Duration and Temperature of Allicin Biosynthesis in Garlic for Inhibiting Bacillus cereus Growth. 

Foodborne illness remains a prevalent issue in today’s society, and B. cereus is a common source. Garlic has been known to demonstrate antimicrobial activity due to the presence of allicin. The biosynthesis of allicin is purported to be highly sensitive to temperature and duration changes due to the alliinase enzyme and the tendency for allicin to quickly convert into secondary compounds. However, investigation into the specific temperature and duration of biosynthesis to maximise allicin production is still insufficient. This study investigated how different biosynthesis durations of allicin measured after the chopping of garlic at 25 °C (77 °F) and 80 °C (176 °F) have affected its inhibitory activity on B. cereus. The Kirby-Bauer Disc Diffusion method was used to measure inhibitory activity by measuring the inhibition zone diameter with a digital calliper. T-Test and ANOVA were used to determine the significance of our results. Our findings indicate the existence of an average experimental optimum biosynthesis duration at 15 minutes for both temperatures and best-fit lines indicating the average optimum to be 12.9 minutes at 25 °C and 13.2 minutes at 80 °C. We also found that garlic preparation at 25 °C is overall more effective than at 80 °C for the prevention of B. cereus growth. Ultimately, an improved understanding of the antimicrobial potential of allicin in garlic can aid in the prevention of foodborne illness.

Introduction

Garlic has been found to have antibacterial properties in food preparation. Every year, an estimated 600 million—approximately 1 in 10 people in the world—fall ill from contaminated food, and 420,000 people die from a foodborne illness¹. The bacterium Bacillus cereus (B. cereus) is a common cause of gastroenteritis and foodborne illness worldwide², with more than 63,000 cases of B. cereus each year in the US³. 

B.cereus is a gram-positive, facultatively anaerobic bacterium ubiquitously found in the environment, soil and food. It is characterised by large rod-shaped cells and is naturally present in a wide range of food products. It can be classified as intestinal, affecting the gastrointestinal system, or non-intestinal, infecting the eyes, respiratory tract and wounds⁴. 

The pathogenicity of intestinal B. cereus is associated with the production of tissue-destructive exoenzymes that produce enterotoxins inside the host after ingestion of the bacteria, inducing fluid accumulation and hemolytic, cytotoxic, dermonecrotic and vascular permeability activities. Ultimately, this results in nausea, vomiting, abdominal cramps; and diarrheal syndromes⁴. The emetic disease is often associated with rice products, potatoes, pasta, and cheese, while diarrheal disease is related to meats, milk, vegetables, and fish. B. cereus can exist as spores, resistant to average cooking temperatures and are only eliminated by high temperatures (121°C for three minutes). Therefore, additional methods to reduce B. cereus presence in foods are needed. 

The benefits of garlic have been widely studied in Western medicine and findings show that garlic supports our immune and digestive systems, lowers cholesterol, and relieves fevers⁵.

The specific preparation of garlic has been purported to affect its antimicrobial properties. Black garlic, a fermented form of garlic, has been alleged to offer double the antimicrobial benefits of regular garlic⁶. In the culinary world, there is the maxim of the “10 Minute Garlic Rule” where, upon chopping, garlic is left 10 minutes before cooking for maximum antibacterial content⁷. However, the former research on the antimicrobial properties of garlic is minimal and additional exploration is warranted to understand its full potential.

Mechanism of action for alliinase and allicin

The antibacterial activity of garlic is due to allicin (S-allyl-cysteine), the major bioactive component of garlic. Allicin is formed by the enzymatic reaction of substrate alliin with the enzyme alliinase.

Alliinase, which is enclosed in a separate cellular compartment from alliin, is only activated once the cell structure is disrupted, allowing it to then interact with alliin. This occurs when garlic is crushed or cut, as it is in food preparation. Alliin is converted into allicin in a two-step reaction: alliinase hydrolyses alliin to allylsulfenic acid and dehydroalanine, and then two molecules of allylsulfenic acid spontaneously condense into one allicin molecule⁸.

After allicin is produced, it is highly unstable at room temperatures and higher, and will soon convert into other compounds such as diallyl sulphide, disulphide and trisulfide which show reduced antibacterial effects compared to allicin¹⁰, as well as dithiins and ajoenes which lack the broad-spectrum antibacterial efficacy of allicin¹¹. Hence, the lifespan of allicin, and thus the period of greatest antibacterial content of garlic, is fairly short-lived. Nonetheless, its specific lifespan has yet to be determined scientifically.

Once activated, allicin disrupts bacterial enzyme function essential for bacterial growth and survival. The specific antimicrobial mechanisms of allicin remain unclear as it involves various modes of action. Mechanistically, however, it is purported that the hydrophobic properties of allicin allow it to readily permeate and damage bacterial cellular membranes. Allicin’s puncture of the outer bacterial membrane destabilises membrane integrity and increases membrane permeability, resulting in cellular content leakage. Allicin’s breach of bacterial cellular compartments causes rapid oxidation of the free thiol groups, such as glutathione and cysteine residues in cellular proteins. A more oxidised glutathione and cysteine pool leads to a higher cellular redox potential and changes to protein cell structure such as through disulfide bond formation¹², resulting in protein misfolding and subsequent loss of function¹³. These proteins, namely alcohol dehydrogenase, thioredoxin reductase, RNA polymerase and DNA gyrase¹⁴, are important in essential metabolic processes for bacteria. Therefore, their loss impairs bacterial growth.

Our study aims to determine how preparation conditions of garlic, specifically biosynthesis duration and common preparation temperatures, affects its antimicrobial content. Temperatures chosen were 25 °C (77 °F) to model room temperature and 80 °C (176 °F) to model the average temperature for low-heat cooking¹⁵. In terms of culinary implications, room temperature is a common storage and preparation condition for many food items, including garlic. Understanding how allicin reacts or degrades at room temperature is vital for maintaining its health benefits during food preparation. Furthermore, garlic is typically cooked in medium-to-low heat conditions through various methods such as roasting, sauteing and grilling¹⁶. Due to lab limitations, 80 °C was also the highest temperature available for testing. 80 °C was thus chosen as a proxy to gauge whether cooking affects allicin content. The biosynthesis duration of allicin was investigated with the ranges of 5, 10, 15, 20 and 25 minutes, as the maxim of garlic biosynthesis duration was 10 minutes⁷ and preliminary trials indicated significant inhibition from 10 to 30 minutes. This interval was also chosen due to time and school lab constraints preventing the measurement of biosynthesis duration per minute.

We hypothesise that there exists an optimal duration of allicin biosynthesis which allows for a maximal zone of bacterial inhibition. We further hypothesise that the optimum duration will be reached quicker but a smaller inhibition zone will be produced at 80 °C (176 °F) than at 25 °C (77 °F). Higher temperatures increase kinetic energy and often catalyse a higher rate of enzymatic reactions¹⁷While higher temperatures may increase the rate of allicin biosynthesis, they may also hasten the degradation of allicin into other compounds. Extreme temperatures also denature alliinase, leading to a decrease in allicin production. Since the optimum temperature of allicin biosynthesis is 35-37 °C, we hypothesise an overall larger bacterial inhibition zone at 25 °C than at 80 °C. 

Considering the prevalence of B. cereus in everyday food consumption, it acts as a relevant test subject to model foodborne illnesses. Determination of the optimum biosynthesis reaction duration of allicin after crushing and juicing garlic on the inhibition of B. cereus, at both normal and high temperatures will provide food preparation insights for the culinary community at large, informing how long garlic, after chopping, should be left and the potential of temperature manipulation to optimize antimicrobial efficacy and reduce incidents of foodborne illness. 

Results

Due to time and school lab constraints, 9 trials were conducted for investigating the effect of duration and temperature of allicin biosynthesis duration.

Effect of biosynthesis duration on antimicrobial activity of allicin

Table 1 shows the change in diameter of inhibition zone due to differing biosynthesis durations of garlic at room temperature 25 °C. On average, 15 minutes was the most effective biosynthesis duration of those tested to maximise the zone of inhibition, with a significant decrease in optimisation for 5-minute intervals (Table 1 and Figure 2), as demonstrated by a greater decrease in the inhibition zone from 15 to 25 minutes compared to 5 to 15 minutes. The lowest percentage error of standard deviation was 8.03% for 29.5mm at 5 minutes, while the highest was 11.7% for 30.6 at 20 minutes (Table 1).

BD / min (± 0.001 sec)	Diameter of inhibition zone / mm (± 0.02mm)
	Trials									Avg	SD	% error of SD
	1	2	3	4	5	6	7	8	9
5	26.0	27.4	26.6	28.3	31.5	31.9	31.5	30.8	31.2	29.5	2.37	8.03
10	35.7	29.2	29.1	32.6	35.4	35.1	30.1	34.5	34.4	32.9	2.73	8.31
15	35.2	35.3	35.0	42.4	33.2	43.1	35.9	36.6	35.1	36.9	3.46	9.38
20	27.4	26.6	26.0	34.5	33.2	33.1	29.9	29.1	35.5	30.6	3.58	11.7
25	18.7	19.3	20.1	21.8	22.1	24.3	22.3	23.0	24.3	21.8	2.03	9.33

Effect of temperature on antimicrobial activity of allicin

Table 2 shows the change in diameter of the inhibition zone due to differing biosynthesis durations of garlic at low-heat cooking temperature (80 °C). Overall, allicin production was lower under 80 °C conditions compared to 25 °C (Table 2 & Figure 3). Specifically, the difference in inhibition zones between the two temperatures was significant at 15 minutes (Figure 3A). Based on each quadratic trendline at 25 and 80 °C, the average optimum duration to maximise the zone of inhibition was 12.9 and 13.1 minutes respectively (Figure 3B), while the average experimental optimum was still 15 minutes at 80 °C (Table 2). The lowest percentage error of standard deviation was 5.97% for 32.6 mm at 15 minutes, while the highest was 9.80% for 20.8 mm at 25 minutes (Table 2).

BD / min (± 0.001 sec)	Diameter of inhibition zone / mm (± 0.02mm)
	Trials									Avg	SD	% error of SD
	1	2	3	4	5	6	7	8	9
5	32.9	28.1	27.7	32.1	25.1	28.3	29.7	25.0	30.3	28.8	2.76	9.58
10	30.2	30.0	34.5	28.3	30.1	30.2	31.8	33.4	36.6	31.7	2.65	8.37
15	32.0	33.8	36.7	29.7	32.1	33.3	31.1	33.0	32.1	32.6	1.95	5.97
20	28.2	26.3	29.2	31.1	34.5	33.0	29.5	32.7	31.3	30.6	2.59	8.44
25	20.2	18.1	17.7	23.1	22.2	20.1	23.4	21.8	20.2	20.8	2.03	9.80

Discussion

B. cereus mediated foodborne illness remains a prevalent problem in our society. The antimicrobial properties of garlic may be able to reduce incidents of B. cereus mediated illnesses. In food preparation practices, our experimental findings suggest that after garlic is chopped, it should be left for approximately 15 minutes before consumption to increase allicin for maximum antibacterial content, or 12.9 minutes at 25 °C and 13.2 minutes at 80 °C based on the best-fit line. Eating garlic raw (25 °C) may also result in slightly higher allicin and thus antibacterial content compared to after cooking (80 °C), however, this difference may only be significant near the optimum duration. Ultimately, these preparation conditions increase food safety through preventing the growth of B. cereus causing foodborne illnesses.

These findings are consistent with the prior literature, including consistency in the allicin-mediated diameters of inhibition zones against B. cereus¹⁸, which speaks to the accuracy of our methodology. Furthermore, the bell-curve nature of the inhibition zones in response to biosynthesis duration is consistent with scientific literature. Initially, more and more alliin is converted to allicin, inhibiting B. cereus growth through membrane damage and thiol group oxidation¹³. After the optimum biosynthesis duration is reached, the conversion of allicin into less effective antimicrobial compounds results in decreased inhibition¹⁰. The optimum temperature of alliinase enzymatic activity is 35-37 °C¹⁷. Consistent with this finding, our results demonstrated greater inhibition at 25 °C compared to 80 °C. Higher temperatures also increase kinetic energy¹⁵, thereby increasing the biosynthesis but also degradation rate, as demonstrated by a slightly shorter average optimum duration of 12.9 minutes at 80 °C and 13.2 minutes at 25 °C based on the best-fit line.

Our study focused mainly on allicin as the active antimicrobial ingredient of garlic. Nonetheless, as the allicin degrades and converts into other compounds, other compounds may still exhibit antimicrobial properties towards B. cereus, but more research is warranted to understand the specific abilities of each secondary compound. Previous studies have shown that antibacterial activity increases with the increase in number of sulfur atoms in the secondary compound, thus suggesting that antibacterial activity is mediated by the formation of disulfide bonds between compounds and bacterial enzymes, ultimately resulting in denaturation due to misfolding¹⁹. In another study, diallyl disulfide, a compound formed by the decomposition of allicin,  showed little remarkable antimicrobial activity unless used in very high concentrations for Escherichia coli and Staphylococcus aureus, with approximately 35× more diallyl disulfides (6.15 mM) needed to inhibit their growth compared to allicin (0.17 mM)¹².

There were several practical limitations to the current study that may have caused low experimental data precision, resulting in high percentage errors. 

 As the experiment took three days to complete, there may have been variations when experimenting on different days in the biological tissue of B. cereus due to being exposed to slightly different conditions, impacting its antimicrobial resistance and thus inhibition zone. Completing trials in one day, frequent monitoring of the bacteria in the shaker, and increased number of trials may eliminate the impact of B. cereus differences on different days. 

Inhibition zones obtained at 10 and 15 minutes were sometimes so large that it was difficult to determine where the diameter ended as it merged with another zone, hindering the accuracy and reliability of our measurements. To improve, one agar plate per disc could be used to measure inhibition.

At 80 °C, potential evaporation effects of garlic juice that may concentrate the garlic juice was not accounted for in our study. Future replications and investigation by placing the agar plate in a container whilst in the water bath is necessary to ensure that the garlic juice concentration remains consistent.

9 trials is a small sample size to generalise our findings. An increased sample size, such as conducting at least 12 trials may decrease the random error of my experimental data and further verify the observed trend.

Allicin degradation beyond the optimum should be measured directly to ensure that the decrease in inhibitory power is not due to potential experimental errors. This can be achieved by quantifying the degradation of allicin by measuring the remaining concentration of allicin using high performance liquid chromatography (HPLC)²⁰.

Our experiments examined the effect of garlic on B. cereus after incubation in a nutrient broth at a temperature of 25 °C and pH of ~6.8, the necessary laboratory conditions which allow for the non-pathogenic bacteria to grow. In reality, the interactions of garlic and B. cereus are more complicated. Though potentially coming into contact when mixed into a prepared dish of food, significant contact may only occur after ingestion of garlic and B. cereus. Conditions within the body, such as stomach conditions of 37 °C and pH of 1.5-3.5 are significantly different from the conditions of our laboratory testing²¹. Further research is needed to determine if the quantity of allicin produced during preparation significantly impacts its ultimate antimicrobial ability in the body. 

Our study only tested 25 °C and 80 °C as they are common preparation and cooking temperatures. Investigation of the biosynthesis duration at the optimum alliinase temperature of 35-37 °C may provide more information into the optimum conditions of garlic preparation. In reality, garlic can also be cooked at higher temperatures or boiled. To provide a more comprehensive understanding of temperature in affecting allicin biosynthesis, higher temperatures, specifically at boiling (100 °C), should also be tested.

For practical purposes, this study utilised a non-pathogenic strain of B. cereus. While still representative of the wild-type, additional research with the pathogenic strain B. cereus is needed to validate our findings in a true wild-type organism. Other bacteria causing food-borne illnesses, namely E. coli and Salmonella, as well as garlic from other geographical regions, can be investigated, assessing whether the same optimum duration effectively works for other pathogens and garlic species. 

Our findings in the present study open the door for more nuanced research to determine the optimal conditions of garlic to reduce foodborne illness. In this investigation, a 15-minute average optimum was observed based on collected data, whilst the best-fit line predicted average optimums of 12.9 for 25 °C and 13.2 minutes for 80 °C. Further analysis using shorter intervals of 1 minute from 10-20 minutes will illuminate a more specific optimum. Testing the antimicrobial activity at time 0 as a baseline will also determine the antibacterial content of directly ingesting garlic after chopping.

Allicin can be stabilised to prevent degradation into other compounds, specifically by producing inclusion complexes with -cyclodextrins and carbamide²². Further investigations using these compounds in combination can be conducted to determine the effectiveness of stabilisation in preserving allicin at varying biosynthesis durations, temperatures and pH. 

Overall our findings have indicated useful potential for allicin in B. cereus prevention in food preparation. Further research will improve understanding of practical applications to prevent foodborne illness. 

Conclusion

Foodborne illness remains a common problem, however, certain foods such as garlic may provide a natural solution. Our findings indicate specific biosynthesis durations of allicin optimise garlic antimicrobial activity on B. cereus at room and low-heat cooking temperatures. We identified the experimental average optimum biosynthesis duration for maximum inhibitory activity to be 15 minutes, and best-fit lines indicated the average optimum to be 12.9 minutes at 25 °C and 13.2 minutes at 80 °C. Biosynthesis at 25 °C is overall more effective than 80 °C at preventing B. cereus growth, although this difference may only be significant at the optimum. These findings suggest the existence of an optimal biosynthesis duration and demonstrate allicin’s short-lived lifespan and the sensitivity of alliinase at temperatures farther from its 35-37 °C optimum. Ultimately, continued investigation of the parameters and potential of garlic as an antimicrobial will contribute to the progress of reducing foodborne illness through natural remedies. 

Methods

Preparation and application of B. cereus 

All experiments were completed using a non-pathogenic strain of B. cereus bacteria (Carolina category number: 154870A). The specific composition of nutrient agar used was 12.5 gm Agar, 5 gm Gelatin peptone, and 3 gm Beef extract. The B. cereus was prepared and applied onto the agar plate containing the nutrient agar using general aseptic techniques. Agar plates were divided into four sections by drawing a cross. 50 ml of B. cereus bacteria was spread onto the agar plate using an L-shape spreader while rotating the dish in a circular motion. 

Preparation, application and measurement of antimicrobial

 As a control, a filter paper disc was dipped into distilled water for 5 seconds and then transferred into one of the four quadrants. The diameter of the filter paper disc will be kept constant at 6 mm for both the control and trials.

Garlic (Allium sativum) imported from China was purchased at the local Welcome grocery store in Hong Kong. The garlic cloves were selected for consistency by choosing fresh garlics determined by a high degree of firmness when squeezed, and were uniform in size and colour. The garlic was peeled into garlic cloves with skin remaining intact. Garlic cloves were juiced via a garlic juicer in order to be applied into the agar plate. The juice was collected in a petri dish up to a marked line of 0.5cm in height to ensure sufficient filter paper dipping depth. The biosynthesis duration began upon juicing the garlic and was left to run for durations of 5, 10, 15, 20 and 25 minutes. This process was done at both 25 °C and 80 °C. To execute this process at 80 °C, the petri dish containing garlic juice was held in an 80 °C hot water bath throughout the biosynthesis reaction. At each of these times, a filter paper disc was dipped entirely into the garlic juice for 5 seconds and transferred onto the agar plate. After antimicrobial application, Parafilm was used to seal all agar plates. The agar plates were then placed into the nutrient agar broth at 25 °C at a pH of 6.8 0.2. After 24 hours, the diameter of inhibition was measured by taking the longest distance observed starting from the edge of the filter paper disc to the inhibited zone using a Vernier digital calliper ( 0.02mm). 

Data analysis

Error bars for each average were calculated using the standard deviation above and below the mean. Percentage of standard deviation was calculated by expressing standard deviation as a percentage of the average of each biosynthesis duration. Both calculations were conducted using the equations feature in Microsoft Excel.

For analysis between two groups and analysis between three or more groups, T-test and one-way ANOVA analyses, respectively, were implemented using Graph Pad Prism 10.3 software. P-value <0.05 was considered significant.

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