Bacillus cereus MHI 86 and Bacillus thuringiensis 5-29 both produce the antilisterial bacteriocin thuricin S – analysis of antimicrobial spectrum, stability, and producer strain suitability, including cytotoxicity evaluation

Food safety relies on a delicate balance between keeping harmful bacteria at bay and ensuring that the methods used to stop them do not introduce new dangers. For decades, scientists have looked to nature for solutions, focusing on a group of substances called bacteriocins. These are tiny proteins made by certain bacteria to fight off their competitors. Think of them as a natural defense system, where one microbe produces a weapon specifically designed to disable another. While many of these substances are well known, the ones produced by the Bacillus family of bacteria are particularly interesting because these microbes are found everywhere, from soil to the food we eat. However, using them as a tool requires more than just finding a bacterium that kills bad germs; researchers must also prove that the producer itself is safe and that the weapon it makes is strong enough to survive the harsh conditions of food processing, such as heat and changes in acidity.

In a recent study, researchers set out to examine two specific strains of bacteria, one from the Bacillus cereus family and another from the closely related Bacillus thuringiensis family. Both were originally isolated from food samples and were suspected of producing a powerful substance known as thuricin S. This substance is famous for its ability to target Listeria monocytogenes, a dangerous pathogen that can survive in refrigerated foods and cause severe illness. The team wanted to know if these two different bacteria were essentially doing the same thing, how they made this weapon, and whether it was safe enough to use in the real world. They did not just look at whether the bacteria killed other germs; they dug deep into the genetic code to see how the instruction manual for making the substance was written, measured the exact size of the protein, and tested how well it held up against heat, acid, and digestive enzymes.

The investigation began by growing the two bacterial strains in a nutrient-rich liquid and watching them over time. The researchers found that both bacteria started producing their antibacterial substance as they entered a rapid growth phase, with the activity becoming clearly detectable after eight hours. One strain, Bacillus cereus MHI 86, showed slightly stronger activity than the other, Bacillus thuringiensis 5–29, but both were effective. When the scientists measured how much of the substance was needed to stop the growth of Listeria, they found that very small amounts were sufficient. The strain from B. cereus required about 5.73 micrograms per milliliter to stop the pathogen, while the B. thuringiensis strain needed about 10.77 micrograms per milliliter. To put this in perspective, a standard antibiotic used for comparison was effective at a concentration of just 0.20 micrograms per milliliter, showing that while the bacteriocin is powerful, it is not as potent as some traditional drugs, though it offers a natural alternative.

To understand how these bacteria built their weapon, the researchers looked at their genetic blueprints. They found that both strains carried a specific set of genes responsible for making thuricin S. These genes were arranged in a repeating pattern, with three copies of the instruction sequence lined up one after another. This arrangement is similar to what has been seen in other related bacteria, suggesting a common evolutionary strategy for producing these substances. The team also checked when these genes were turned on. They found that the bacteria consistently produced the genetic instructions for the bacteriocin starting from eight hours of growth, which matched the timing of when the antibacterial activity was first observed. This confirmed that the bacteria were actively manufacturing the substance during their growth phase, rather than just storing it.

Next, the scientists needed to confirm exactly what the substance looked like and how heavy it was. They purified the substance from the liquid where the bacteria grew and ran it through a series of tests. One test, which separates proteins by size, showed a distinct band at approximately 3.1 kilodaltons, a unit used to measure the mass of molecules. To get a more precise measurement, they used a high-tech machine that weighs individual molecules. This analysis revealed that the main active substance had a mass of 3137.6 daltons. This specific weight matched the known profile of thuricin S, confirming that both bacteria were indeed producing the same type of weapon. They also found a second, slightly smaller version of the substance, which might be a variant or a slightly altered form, but the primary active ingredient was clearly identified.

The true test for any food preservative is whether it can survive the conditions it will face in the real world. The researchers subjected the purified substance to various stresses. They found that it remained fully active in acidic and neutral environments, working well at pH levels as low as 3 and as high as 7. This is crucial because many foods, such as fermented meats and cheeses, are acidic. They also tested its resistance to heat. The substance retained over 89 percent of its killing power even after being heated to 80 degrees Celsius, and it still held onto 87 percent of its activity after being boiled at 100 degrees Celsius. However, if the temperature was raised to the extreme levels used in sterilization, around 121 degrees Celsius, the activity dropped significantly. This suggests that while the substance is tough, it is not indestructible and might be best used in foods that are not subjected to the highest sterilization temperatures.

The researchers also checked how the substance reacted to enzymes, which are the proteins in our bodies and in food that break down other proteins. When they treated the substance with enzymes like trypsin and chymotrypsin, which are common in digestion, it remained largely active. However, when they used a very strong enzyme called proteinase K, the substance was completely destroyed. This result confirmed that the weapon is indeed a protein, as it was broken down by the enzyme designed to eat proteins, but it was robust enough to withstand the milder enzymes it might encounter in food or the human gut.

Perhaps the most critical part of the study was ensuring that the bacteria producing this weapon were safe to use. Bacillus cereus is a species that can sometimes cause food poisoning, so the researchers had to be certain these specific strains did not produce harmful toxins. They tested the bacteria for the production of non-hemolytic enterotoxin, a toxin associated with diarrhea, and found that neither strain produced any detectable amounts of it, even under conditions that usually encourage toxin production. Furthermore, they tested the safety of the bacterial liquid on human intestinal cells grown in a lab. The liquid from both strains caused no damage to the cells, whereas liquid from a known toxic strain caused significant harm. This safety profile is essential, as it means the bacteria can be used to protect food without introducing new health risks.

The study concludes that both Bacillus cereus MHI 86 and Bacillus thuringiensis 5–29 are excellent candidates for producing thuricin S. They generate a potent, stable, and safe substance that can effectively target Listeria and other harmful bacteria. Because the substance can withstand heat and acidity, it holds promise for use in preserving foods, particularly those that are pasteurized but might be contaminated later during processing. The research provides a clear picture of how these bacteria make their weapon, how the weapon works, and why it is safe to use, offering a potential new tool for keeping our food supply safer without relying solely on traditional chemical preservatives.