Probiotic Bacillus as fermentation agents: Status, potential insights, and future perspectives

Highlights

• •Probiotic Bacillus are mostly found in solid-state fermented foods.
• •It can provide better flavor and nutritional value to fermented foods.
• •Fewer and less systematic studies on probiotic Bacillus as fermentation agents.
• •Probiotic Bacillus have the potential to be the next generation food fermentation agents.

Abstract

Probiotic Bacillus strains can solve the problems of single flavor and long fermentation time of fermented products caused by the lack of certain functional genes and insufficient metabolism ability of fermenter strains (Lactobacillus and Bifidobacterium) at the present stage. There is a lack of systematic evaluation and review of probiotic Bacillus as food fermentation agents. In this paper, it is observed that probiotic Bacillus strains are involved to varying degrees in liquid-state, semi-solid state, and solid-state fermentation and are widely present in solid-state fermented foods. Probiotic Bacillus strains not only produce abundant proteases and lipases, but also effective antifungal lipopeptides and extracellular polymers, thus enhancing the flavor, nutritional value and safety of fermented foods. Bacillus with probiotic qualities is an underutilized group of probiotic food fermentation agents, which give a potential for the development of fermentation technology in the food business and the integration of ancient traditional fermentation techniques.

Introduction

Fermented foods, with a history spanning thousands of years, constitute a unique and integral part of global culinary cultures (Mukherjee, Breselge, Dimidi, Marco, & Cotter, 2023). Fermentation serves as an effective means for both food production and preservation. Fermented food is obtained after microbial reproduction and metabolism of raw food materials under natural conditions or through the addition of microbial agents. It has a high nutritional value and a unique flavor, is safe, and has high long-term preservation potential (Estruch and Lamuela-Raventós, 2023, Zhang et al., 2023, Zhang et al., 2023, Zhang et al., 2023, Zhang et al., 2023). During the fermentation process, various microorganisms change the chemical makeup of raw materials, improving the nutritional content of fermented food and providing customers with health advantages (Ashagrie et al., 2023, Louw et al., 2023). Based on differences in fermentation processes, fermentation can be broadly categorized into liquid-state fermentation, semi-solid fermentation, and solid-state fermentation. Examples of typical liquid-state fermented foods include fermented dairy products (Tian, Xiong, Yu, Chen, & Lou, 2023) and fermented fruit juice beverages (Zhang et al., 2023, Zhang et al., 2023, Zhang et al., 2023, Zhang et al., 2023). Semi-solid fermented foods encompass fermented vegetable products (Torres, Verón, Contreras, & Isla, 2020) and fermented fruit products (Li et al., 2023, Li et al., 2023, Li et al., 2023). Solid-state fermented foods predominantly include fermented cereal and legume products (Lingua et al., 2022, Xie et al., 2019), fermented tea products, and fermented meat products (Ojha, Kerry, Duffy, Beresford, & Tiwari, 2015).

Probiotics play an important function in the fermentation process of food, producing taste components and ensuring the quality and safety of fermented foods (Zhang et al., 2023, Zhang et al., 2023, Zhang et al., 2023, Zhang et al., 2023). Currently, the exploration and research of probiotic strains is predominantly focused on Lactobacillus and Bifidobacterium, which are globally recognized probiotic genera (Gaur and Gänzle, 2023, Li et al., 2023, Li et al., 2023, Li et al., 2023). There are also relevant studies on bacteria such as Staphylococcus and Streptococcus, as well as yeast and fungi such as black mold (Guidi et al., 2023, Jans et al., 2017, Khusro and Aarti, 2022). However, in traditional fermented foods, the natural fermentation process benefits from the huge macrogenome and metabolome of a complex microbiota. This leads to the formation of unique qualities and rich flavors, a process that is not accomplished by solely lactic acid bacteria or a few specific strains (Rodzi & Lee, 2021). The use of a single fermentation agent often results in long fermentation times and inferior flavors. The development of different composite fermentation strains is a trend in the industrialized production of fermented foods. It is important for the construction of quality and flavor in fermented foods, as well as safety regulation (Luo et al., 2023). Therefore, in-depth and continuous exploration of probiotic strains with good fermentation performance is necessary for the industrialized development of traditional fermented foods (Saarela, 2019). See (Fig. 1).

Fig. 1.

Overview of probiotic Bacillus strains as next-generation Fermentation Agents.

In addition to the popular probiotic strains mentioned above, most Bacillus spp. exhibit probiotic activity. In recent years, a variety of probiotic Bacillus strains, including Bacillus coagulans, Bacillus licheniformis, Bacillus subtilis, Bacillus velezensis and Bacillus clausii, have been tested in vitro and in vivo for possible probiotic activities. Probiotic Bacillus strains have been shown to produce a variety of enzymes, including cellulases, amylases, proteases and lipases. Furthermore, they can produce antimicrobial metabolites, such as bacteriocins and peptides, which hinder the growth and reproduction of dangerous bacteria (Soares et al., 2023, Zhao et al., 2023). Probiotic Bacillus strains can solve the problems of single flavor and long fermentation time of fermented products caused by the lack of certain functional genes and insufficient metabolism ability of fermenter strains (Lactobacillus and Bifidobacterium) at the present stage, and also have great potential in improving the safety of fermented products (Shan et al., 2023). Although a great number of relevant studies have reported the presence of Bacillus spp. in fermented foods, their importance in sustaining human and animal health has been acknowledged. Probiotic Bacillus strains, in contrast to lactic acid bacteria, have received less attention in the fermented food sector and are not widely popular among producers and consumers. (Elshaghabee et al., 2017, Tamang et al., 2016). Therefore, to promote probiotic Bacillus strains as food fermentation agents, it is critical to understand the distribution of probiotic Bacillus in various fermented foods, elucidate the potential and benefits of probiotic Bacillus strains as fermentation agents, and determine their primary contribution to fermented foods.

In the present paper, we examine the distribution of probiotic Bacillus in the microbial composition of traditional natural fermented foods that have a long history of safe consumption, as well as recent advances in the research of the probiotic qualities of Bacillus as fermentation agents. Furthermore, the development prospects of probiotic Bacillus in the modernization of the fermentation industry, as well as the challenges of fermented food production, are examined in order to provide a reference for future research on next-generation food fermentation agents.

Current status of research on probiotic Bacillus in fermented foods

The position of probiotic Bacillus in the microbial composition of fermented foods

Liquid-state fermented foods

Liquid-state fermented foods mainly include fermented dairy products and fermented fruit juice. Liquid-state fermented foods are commonly produced by single fermentation or co-fermentation of Lactobacillus, Bifidobacterium, and some yeasts (Ilango & Antony, 2021). In addition, the presence of probiotic Bacillus spp. has been documented in a limited number of naturally occurring liquid-state fermented foods, as indicated in Table 1.

Table 1.

Analysis of the composition of probiotic Bacillus in the microbiome of traditional fermented foods.

Species	Product category	Product name/ Country	Raw material	Identified Probiotic Bacillus strain(s)	Reference
Liquid-state fermented food	Fermented dairy products	Thai milk kefir/Thailand	Milk, water	B. amyloliquefaciens SD-32, Bacillus sp. LB15, Bacillus sp. LD12AP, Bacillus sp. C87, and B. methylotrophicus	(Vijitra & Sirirat, 2016)
		Kishk/Egypt	Wheat, milk	B.subtilis	(Blandino et al., 2003)
		Dahi/Pakistan	Milk	B.cereus, B. licheniformis, B. mycoides and B. subtilis	(Khan et al., 2023)
	Fermented fruit juice	Rhizome juice of zingiber officinale/Korea	Zingiber officinale	B.fungorum and B. subtilis	(Blandino et al., 2003)
		Tepache and garapiña/Mexico	Pineapple	B.mexicanus and B. subtilis	(Karina et al., 2021)
		Fermented coconut water/Southeast Asia	Fresh coconut water	B. velezensis FCW2	(Raj et al., 2023)
		Fermented red dragon fruit drink/Malaysia	Fresh red dragon fruits(Hylocereus polyrhizus)	B.tequilensis and B. subtilis	(Lim et al., 2023)
Semi-solid fermented odfo	Fermented vegetable products	Kimchi/Korea	Mustard leaf	B. inaquosorum	(Kook et al., 2019)
		Gochujang/Korea	Chili powder, glutinous rice powder, soy porridge with salt, flavorings, for instance, shallots and garlic, as well as sweetener in the form of sugar syrup	B. velezencis	(Jang et al., 2011)
		Fermented brine pickle/West coastal districts of India	Mangos, cabbages, Chili powder, fish sauce	B. Licheniformis KT921419, B. amyloliquefaciens KT921420, B. subtilis KT921421, B. methylotrophicus KT921422, B. safensis KT921423and B. licheniformis KT921424	(Ragul et al., 2020)
		Soibum/ India	Bamboo shoot	B.subtilis, B. lichniformis, B. coagulans, B. cereus, B. pumilus	(Tamang et al., 2017)
		Rhujuk/ Bastanga, india	Bamboo shoot	B.subtilis and B. licheniformis	(Jamir & Deb, 2021)
		Agbelima/ West Africa	Cassava	B.subtilis, B. licheniformis, and B. pumilus	(Obilie et al., 2003)
		Tape/ Indonesia	Cassava	B.subtilis, B. amyloliquefacie and B.thuringiensis	(Barus et al., 2013)
		Torshi/ Iran	Green pepper, Green cabbage, Red Cabbage, Cauliflower, Carrot, garlic, Celery and Persian shallot	B. safensis 437F, B. atrophaeus 1630F and B. amyloliquefaciens 1020G	(Talebi et al., 2018)
		Ntoba Mbodi/ Congo	Cassava leaves	B.subtilis, B. licheniformis, B.amyloliquefaciens, B. pumilis, B. sphaericus and B. xylanilyticus	(Mbozo et al., 2017)
		Fermented pickle/ China	Cabbage and NaCl	Bacillariophyta	(Sun et al., 2022)
		Pickles/ Egypt	Fruits and vegetables, NaCl	B.acidicola BPS4, B. amyloliquifaciens BPS20, B.mycoides BPS33	(Enan & E.F., M., Abdel-Haliem, & Tartour, E. , 2014)
	Fermented fruit products	Panchamirtham/ India	Banana, brown sugar, seedless dates, sugar candy, honey, cardamom and ghee	B.valezensis M4S1B1, B. safensis M5S2B8 and Bacillus sp. M7S2B9	(Maheshwari et al., 2019)
Solid-state fermented food	Fermented cereal and legume products	Daqu/ China	Grains (wheat, rice, sorghum, and barley)	B.subtilis, B.amyloliquefaciens, B. velezensis and B.licheniformis	(Zhu et al., 2023)
		wheat Qu/ China	Wheat	Bacillus	(Peng et al., 2023)
		Soy sauce/ Korean	Soybean	B.amyloliquefaciens	(Lee et al., 2017)
		Koji/ China	Soybeans, wheat flour	B.amyloliquefaciens, B. subtilis, B.lincheniformis, B. methylotrophicus, B.aerius, B. halmapalus, B. flexus, B.thuringiensis and B. coagulans	(Zhang, Xiong, et al. 2023)
		Natto/ Japan	Soybean	B. subtilis, B. natto	(Dong et al., 2020)
		Tungrymbai and bekang/ India	Soybean	B. licheniformis, B. pumilus B.subtilis and B. coagulans	(Chettri & Tamang, 2015)
		Cheonggukjang/ Korean	Soybean	B.thermoamylovorans, B. licheniformis, B. glycinifermentans, B. subtilis, B. paralicheniformi	(Tamang et al., 2022)
		Doenjang-meju, Korean	Soybean	B. velezensis	(Han et al., 2023)
	Fermented tea products	Post-fermented tea/ China	Fresh leaves or mature shoots of tea plants (Camellia sinensis var. sinensis)	B.subtilis DTM01, B.licheniformis DTM06, B. laterosporus DTM03, B. coagulans DTM02, B. pumilus DTM04	(Zhao, Lou, et al. 2021)
		Fu brick tea/ China	Pu-erh tea	B.subtilis	(Li et al., 2022)
	Fermented meat products	Androlla and Botillo / Spanish	pork, salt	B. amyloliquefaciens SA35 and B. subtilis SB07	(Cachaldora et al., 2014)
		Salame di Senise/Southern Italian	Pork, senise, wild fennel, garlic and salt	B. subtilis tr53, tr50 and trf22	(Baruzzi et al., 2005)
		Pla-ra/ Thailand	Marine fish, salt, rice bran	B. subtilis subsp Subtilis UD6-2	(Thongsomboon et al., 2023)
		Fermented cassava fish/ Benin	Cassava fish	B. subtilis, B.licheniformis, B. megaterium, B. mycoides, and B. cereus	(Anihouvi et al., 2007)
		Fish Sauce/ China	Fish (sardine, anchovy, and menhaden, among others) and sea salt	B.subtilis, B. amyloliquefaciens, B. aryabhattai, B. vallismortis, B. cereus, B. megaterium, B.tequilensis, B.licheniformis, B. marisflavi, B. methylotrophicus, B. vietnamensis	(Xiao et al., 2014)
		Utonga-kupsu, Hentak and Ngar/ North-East India	Esomus danricus, Puntius sophore, Amblypharyngodon mola, Channa punctata, Mystus vittatus, Puntius sophore, etc.	B. subtilis	(Singh, Mandal, Lalnunmawii, & Kumar, 2018)
		Ngari/ Manipur in India	Sun-dried small cyprinid fish Puntius sophore Ham.	B. indicus	(Devi et al., 2015)
		Ka-pi / Thai	Shrimp	salacetis spp. Bacillus species	(Nakamura et al., 2022, Yiamsombut et al., 2021)

Fermentation processes can change the enzymatic activity of raw materials and the metabolic activity of microbes, influencing the nutritional and bioactive qualities of the food matrix, which can have good impacts on human health (Mukherjee et al., 2023). Many scientific research have established that fermented dairy products have antihypertensive properties, improve systemic immunity, and reduce cholesterol and blood pressure. They can also be a rich source of bioactive peptides generated during protein hydrolysis, with various potential health advantages for the endocrine, digestive, cardiovascular, immunological, and neurological systems (Companys et al., 2020). It is worth mentioning that Thai Milk kefir, a stirred and fermented milk, has anti-inflammatory, immunomodulatory, antimicrobial, antiproliferative, antimutagenic, and anticarcinogenic properties and has the potential to become a functional food. To date, many studies have been published on different bacteria and yeasts isolated from kefir from different parts of the world (Urdaneta et al., 2007). Vijitra & Sirirat (Vijitra & Sirirat, 2016) found only Bacillus spp. in the microbial composition of Thai Milk kefir, mainly B. amyloliquefaciens, which and produce extracellular polysaccharides on glucose, lactose, and sucrose. In Egypt, Kishk is usually made by combining strained yogurt with milled dry wheat (cracked and gluten-free steamed grain wheat) and allowing it to ferment at room temperature for varied amounts of time. The milk is fermented, and the resulting paste is dried to a moisture content of 10 %-13 % before grinding into powder. The substance is preserved in the form of dried brown balls with a rough surface and a firm feel. The bacteria responsible for fermentation include Lactobacillus plantarum, Lactobacillus casei, B. subtilis, and Saccharomyces cerevisiae (Blandino, Al-Aseeri, Pandiella, Cantero, & Webb, 2003). Dahi is a popular fermented dairy product in Pakistan. It is made by bringing room temperature boiled or buffalo milk to room temperature, adding artisanal cultures (obtained by a reverse tilting technique), and then letting it sit for at least one night (sometimes up to four days, depending on the season) until a curdled product forms. Strains of Lactococcus lactis, Lactobacillus casei, Streptococcus thermophilus, and Lactobacillus bulgaricus, as well as probiotic Bacillus species such B. cereus, B. licheniformis, B. mycoides, and B. subtilis, are among the fermentation microorganisms found in Dahi (Khan, Bashir, & Imran, 2023).

Fermented juices, obtained by fermentation of fresh fruits with bacterial strains, have a unique flavor and high nutritional value (Zhang, Hong, et al. 2023). In recent years, studies on probiotic Bacillus spp. in fermented fruit juices have been conducted. Bacillus fungorum, B. subtilis, and others isolated from the juice of Zingiber officinale rhizome in Korea were recognized as potential probiotics (Sathiyaseelan, Saravanakumar, Han, Naveen, & Wang, 2022). Garapiña, a traditional Mexican fermented juice, is made from pineapple pulp and rind, whereas tepache is usually made from pineapple rind only; however, it can be made from other fruits such as apple, orange, and guava as well. The drink is produced by inducing fermentation of pineapple peel and brown sugar in water, which does not require pre-inoculation. The ingredients are placed in wooden barrels called “tepacheras,” where the pineapple peel and pulp ferment spontaneously for three days. Afterwards, a sweet, refreshing, and pleasant drink is obtained, thanks to microorganisms such as Weissella confusa, B. graveolens, B. mexicanus, and B. subtilis (Karina et al., 2021). Fermented coconut water, a functional beverage fermented with beneficial microorganisms from fresh coconut water, is consumed in a variety of regions, particularly Southeast Asia, due to its refreshing taste and potential health benefits. It contains fewer calories and sugar, making it a healthier alternative to soft drinks and sugary beverages. Raj et al. (Raj, Suryavanshi, Kandaswamy, Ramasamy, & James, 2023) investigated the probiotic potential of B. velezensis FCW2 isolated from spontaneously fermented coconut water using in vitro and genomic characterisation. Lim et al. (Lim et al., 2023) used molecular methods to determine the variety of local microorganisms involved in the spontaneous fermentation of red dragon fruit drink. From the fermented red dragon fruit drink, twenty bacterial cultures were discovered, primarily Klebsiella pneumonia, Klebsiella pneumonia, Brevibacillus parabrevis, B. tequilensis and B. subtilis.

Semi-solid fermented foods

Semi-solid fermented foods, such as pickled vegetables and fruits, represent a typical food category with a long history of consumption worldwide. Indeed, varied individuals from various cultures are drawn to these foods because of their appealing sensory qualities, high nutritional value, and long shelf life. The extensive history of fermentation, with many products originating from natural fermentation processes, makes it an intriguing source for isolating novel probiotic strains. The discovery and identification of Bacillus strains with significant probiotic potential will enhance their contributions to the food industry (Behera, Sheikha, Hammami, & Kumar, 2020).

Kimchi is a traditional fermented Korean food made through the fermentation of vegetables. Due to its delightful taste and beneficial effects on human health, it has become a globally popular food. Kook et al. (Kook, Lee, Jeong, & Kim, 2019) isolated B. licheniformis BioE-BL11 and Enterococcus faecium LMD18 from Kimchi. These strains produced a substantial amount of extracellular polysaccharides. Additionally, Jang et al. (Jang, Kim, Park, & Park, 2011) conducted a microbial community analysis of Gochujang, another traditional Korean fermented food. They identified a total of 31 microorganisms, with B. velezensis accounting for 29 % of the population, making it the predominant microbe in Gochujang.

B. licheniformis KT921419, B. amyloliquefaciens KT921420 and B. subtilis KT921421 were identified from the traditional fermented brine mango pickle in the western coastal region of India. They have substantial antioxidant, antidiabetic, and antityrosinase activities. These strains can serve as novel fermentation agents or co-cultures imparting health benefits in food systems. Furthermore, the lengthy history of pickle consumption attests to the safety of these strains, and their additional health-promoting properties make them an ideal candidate for marketing pickles as healthy and functional foods (Ragul, Kandasamy, Devi, & Shetty, 2020). Soibum is an ethnic fermented bamboo shoot dish from Manipur, India. Thin slices of immature shoots are packed securely in an enclosure, sealed with a polyethylene film on top, and pressed with an appropriate weight. During fermentation, the bottom of the fermentation chamber is punctured to drain acidic fermentation juice, which is permitted to ferment for 6–12 months. Fermentation's microbial population includes lactic acid bacteria such as Lactobacillus plantarum, Lactobacillus brevis, Lactobacillus coryniformis, and Lactobacillus delbrueckii, as well as Bacillus spp. including B. subtilis, B. licheniformis, B. coagulans, B. cereus and B. pumilus (Tamang, Holzapfel, Shin, & Felis, 2017). Rhujuk, an indigenous fermented cuisine from Nagaland, India, has been discovered to have Bacillus spp. as the main bacterium in majority of the foods based on a combination of morphological and genetic investigations. Agbelima is an African sourdough product. It is mainly made from heap-fermented cassava roots to reduce the rubbery texture of the subsequently produced flour (Jamir & Deb, 2021). Obilie et al. (Obilie, Tano-Debrah, & Amoa-Awua, 2003) demonstrated that several fungi and Bacillus spp. isolated from heap-fermented cassava roots were able to break down cassava tissues. Tape is a traditional cassava fermented dish in Indonesia, and its quality is dictated by the microorganisms engaged in the fermentation process, with the amylase-producing Bacillus spp. (B. subtilis, B. amyloliquefaciens and B. thuringiensis) dictating the quality of cassava (Barus, Kristani, & Yulandi, 2013).

Torshi, a pickled vegetable condiment from the Middle East. Typically, it is made by households rather than industries, and there are hundreds of recipes depending on local traditions. Torshi is produced in eastern Iran using a variety of vegetables, including green peppers, green kale, red kale, cauliflower, carrots, garlic, celery, and Persian green onions, which are pickled in salted vinegar for several weeks. Three novel Bacillus strains, B. safensis 437F, B. atrophaeus 1630F, and B. amyloliquefaciens 1020G, were identified as promising probiotics (Talebi, Makhdoumi, Bahreini, Matin, & Moradi, 2018).

Popular fermented food from the Republic of the Congo called Ntoba Mbodi is a major source of protein for the diets of the local populace. It is made by gathering cassava leaves, letting them wither for 2–3 days, and then cleaning, chopping, washing, dividing, and wrapping the leaves in huge leaves like papaya or Senegalese plant leaves. The combination is then allowed to ferment at room temperature for two to four days. Bacillus species, such as B. subtilis, B. licheniformis, B. amyloliquefaciens, B. pumilus, B. cereus, and B. xylosus, are the main microbes that cause fermentation in these mixes. B. subtilis is typically described as the dominating species (Mbozo et al., 2017).

Fermented pickle is a Chinese traditional fermented food. To produce fermented pickle, vegetables are immersed in a salt solution and undergo lactic acid fermentation. Pickles fermented by Lactobacillus not only have a delicious taste and high nutritional value, but also offer digestive assistance and potential benefits in preventing atherosclerosis. Lactic acid bacteria are the major microorganisms in pickles and play an important role in taste creation (Sun et al., 2022). Additionally, several research imply that non-lactic acid bacteria also play a major role in flavor generation during the vegetable fermentation. Throughout the natural fermentation and storage of pickles, there is a diverse microbial community, and the complex succession of microbial colonies and diverse metabolic pathways significantly influences flavor formation. Bacillus and Streptococcus bacteria play a role in flavor creation throughout this process. Enan and colleagues (Enan & E.F., M., Abdel-Haliem, & Tartour, E. , 2014) recovered 61 strains of bacteria from Egyptian pickles and discovered Lactobacillus plantarum LPS10, B. acidicola BPS4, B. amyloliquefaciens BPS20, and B. mycoides BPS33 as possible probiotic strains.

Panchamirtham is a traditional fermented fruit mixture (banana, jaggery, seedless dates, sugar candy, honey, cardamom, and ghee) which is widely consumed in the southern regions of India. This traditional fruit mixture is rich in dietary fiber, vitamins, proteins, and minerals. Panchamirtham contributes to resistance against infectious pathogens in the body and serves as a potent antioxidant, providing a source of minerals, vitamins, and carbohydrates. Maheshwari et al. (Maheshwari et al., 2019) conducted 16S rRNA sequencing and evaluated the microbiota in Panchamirtham, identifying Bacillus spp. with probiotic potential such as B. safensis M5S2B8 and B. velezensis M4S1B1.

Solid-state fermented foods

Solid-state fermented foods encompass a variety of types, including cereal- and legume-based fermented products, fermented tea products, and fermented meat products. Extensive research data indicate that these fermented foods, often produced in open environments, have a diverse and complex microbial composition. In addition to common Lactobacillus, the types and quantities of probiotic Bacillus strains in these foods are diverse and intricate (Li, Zheng, et al. 2023).

Typical representatives of cereal and soybean fermented products include Chinese liquor (Baijiu) and soy sauce, which probiotic Bacillus strains are detected more frequently and play crucial role in the fermentation process, contributing substantially to the quality and flavor of the products. Chinese liquor, with its rich history and cultural significance, has gained widespread popularity globally. In recent years, its market dominance has grown, with a total production of 7,156,000 kiloliters and a profit of 170.194 billion yuan in 2021. Chinese liquor has emerged as a vital business that contributes to local economic development. Brewing techniques in each region are always evolving and improving, resulting in distinct microbiological ecosystems and flavor variations. Despite regional differences, the production of Daqu (a large fermentation starter) has consistently been a core process in Chinese liquor production. The Chinese liquor industry has been based on the principle of “First make Daqu, then brew” for thousands of years (Pan et al., 2023, Zhang et al., 2023). According to research, barley malt can introduce microorganisms for stacked fermentation while also providing taste ingredients for the liquor. Typical raw materials used are barley, wheat, and pea, which are high in starch and protein.

These macromolecules are degraded by a variety of enzymes, including amylases, glucoamylases, and proteases (typically acidic proteases), which provide nutrients and substrates for microbial growth and taste metabolism. Esterase is also an important enzyme involved in the creation and hydrolysis of volatile esters, which are essential for flavor development. These enzymes are primarily produced by microbes such as Aspergillus, Rhizopus, Bacillus spp., Lactobacillus and Saccharomyces cerevisiae. Related studies found that Bacillus was also the dominant genus of Daqu (Peng et al., 2023, Zhu et al., 2023). The fermentation process is driven by the functional microbiota, which also effects the flavor and end result. Soy sauce, a dark and salty condiment, is produced through the fermentation of cooked soybeans and roasted grains. It is a fundamental component in Chinese, Japanese, and other Asian cuisines, gaining popularity in Western cuisines as well. Soy sauce is often produced by soaking soybeans in water, heating them, and blending them with roasted wheat flour. The mixture is then injected with Aspergillus oryzae or Aspergillus sojae and cultured for 2–3 days at 25–30 °C to produce koji. The koji is then mixed with a high-concentration salt solution to achieve a final salt concentration of 22 %–23 %, and left to ferment at room temperature for 6–12 months. China produces approximately 5 million tons of soy sauce annually, accounting for over 50 % of the world's production. The main microorganisms involved in soy sauce fermentation are Bacillus spp., such as B. licheniformis, B. velezensis, B. amyloliquefaciens, B. methylotrophicus, B. aerius, B. halmapalus, B. flexus and B. thuringiensis (Lee et al., 2017, Zhang et al., 2023).

Additionally, probiotic Bacillus spp. is the main bacteria in many viscous fermented foods in Asian. Natto, typically made from soybeans fermented by B. subtilis and B. natto, is a traditional Japanese fermented food. Natto has a golden color and a distinctive aroma and forms a white, transparent bacterial film on the surface. When stirred, it exhibits long and slimy strands (Dong et al., 2020). In India's Meghalaya state, tungrymbai and bekang is a naturally fermented soybean sticky food consumed as a side dish. The microbial species include B. thermoamylovorans, as well as B. licheniformis, B. glycinifermentans, B. subtilis and B. paralicheniformis (Chettri & Tamang, 2015). Cheonggukjang, a traditional fermented soybean food from Korea, is produced by fermenting steamed soybeans with natural microbial cultures. The main microbes in Cheonggukjang are B. thermoamylovorans and B. licheniformis (Tamang et al., 2022). Doenjang, another ancient fermented soybean dish from Korea, has been taken for millennia as both a protein source and a flavor, comparable to miso in Japan and tempeh in Indonesia. Traditionally, Doenjang is made by mixing and fermenting brine with moldy cooked soybeans, in which naturally transported bacteria destroy soy proteins and produce a variety of nutritious chemicals. Bacillus is believed to be dominant in soybean paste, and the probiotic Bacillus spp. involved in its fermentation are B. licheniformis and B. subtilis (Han, Baek, Chun, & Jeon, 2023).

Post-fermented tea (also known as black tea) is a distinctive Chinese tea product created from the tea plant's fresh leaves or mature buds. The manufacturing process consists mostly of enzyme inactivation, rolling, heap fermentation, steaming, and drying. Yunnan Pu-erh Tea, Hubei Qingzhuan Tea, Hunan Fuzhuan Brick Tea, and Guangxi Liubao Tea are the four most well-known post-fermented teas, which have received widespread attention from tea consumers in Hong Kong, Macau, mainland China, Southeast Asia, and other countries for their unique flavor quality and health benefits (Zhu et al., 2020). During the heap fermentation stage, tea is turned over on a weekly basis, and the fermentation is ended when the tea blocks turn reddish-brown and no longer taste bitter. This natural solid-state fermentation process involves a number of events, including degradation, oxidation, condensation, structural alteration, methylation, and glycosylation. Previous research has shown that the concentrations of catechin derivatives, flavonoids and their glycosides, phenolic acids, alkaloids, and terpenoids fluctuated dramatically during solid state fermentation. The post-fermentation process takes place in a very complex microecosystem involving bacteria and fungi (Lin et al., 2021). Antagonism between microorganisms in this microecosystem has piqued interest, and the potential antifungal capacity of Bacillus strains in particular has been extensively researched due to their ability to produce a wide range of potent compounds that inhibit fungal growth and mycotoxin production. Bacillus spp., including B. subtilis, B. licheniformis, B. laterosporus, and B. coagulans, produce tiny antifungal peptides with molecular weights of ≤ 10 kDa (Li et al., 2022, Zhao et al., 2021).

Typically, fermentation can occur spontaneously due to natural microbial contamination of the raw materials. Fermentation is an ancient technique of preserving food, and the complicated microbial makeup during fermentation provides distinctive aromas, textures, and nutritional value to meat products (Cruxen, et al., 2019). Fermented meat products include pork, beef, lamb, and fish, as well as river shrimp. Fermented meat products are classified into two types: those produced from whole or sliced meat (cubes and jerky) and those prepared with minced meat. Lactic acid bacteria are the most common microbes involved in meat fermentation, followed by coagulase-negative Staphylococcus, Micrococcus, and Bacillus spp., such as B. subtilis, B. mycoides, B. lentus, and B. amyloliquefaciens. Fermented fish and fish products are part of many people's diets worldwide. According to tradition, they are eaten as major dishes, side dishes, and sauces. Several bacterial and yeast species have been identified in fermented and traditionally cured fish items around the world, including probiotic Bacillus spp., mainly B. subtilis, and B. amyloliquefaciens. Cachaldora et al. (Cachaldora, Fonseca, Gómez, Franco, & Carballo, 2014) investigated the growth and metabolic properties of 19 Bacillus species isolated from Androlla and Botillo sausages, discovering that B. amyloliquefaciens SA35 (isolated from Androlla) and B. subtilis SB07 (isolated from Botillo) could hydrolyze myoplasmic proteins and myosin, as well as have lipolytic activity (very high activity in SB07 and high activity in SA35). Both strains showed low levels of decarboxylase activity. These findings imply that Bacillus strains may have a role in the maturation and development of sensory qualities in these sausages. The results of the technological properties of Bacillus strains (B. subtilis tr53, tr50 and trf22) isolated from the southern Italian sausage “Salame di Senise” by Baruzzi et al. (Baruzzi, Matarante, Caputo, & Morea, 2005) showed that B. subtilis strains always present in meat curing can contribute to the development of texture and organoleptic properties. Strains that are constantly present in meat curing could influence the texture and organoleptic properties of sausages.

Fermented fish products are consumed in many nations throughout the world. According to tradition, they are eaten as main courses, side dishes, and as condiments. Several species of bacteria and yeasts have been documented to be present in fermented and traditionally cured fish products across the globe (Belleggia & Osimani, 2023), including probiotic Bacillus species mainly B. subtilis, B. licheniformis, B. megaterium, B. mycoides and B. cereus. Traditional Thai salt-fermented freshwater fish (Pla-ra) is a popular cuisine produced and consumed throughout Thailand. The Thai Food and Drug Administration has authorized a local fermenter, a combination of B. subtilis UD6-2 and Virgibacillus halodenitrificans NCFF-2, as an acceptable food culture for Pla-ra fermentation (Thongsomboon et al., 2023). Anihouvi et al. (Anihouvi, Sakyi-Dawson, Ayernor, & Hounhouigan, 2007) investigated the microbial alterations that occurred during the spontaneous fermentation of cassava fish and discovered that the fermented fish's microbial community included a diverse variety of Gram-positive bacteria (92.7 %) and Gram-negative bacteria (7.3 %). Bacillus spp. (48.7 %), Staphylococcus spp. (27.3 %), and Micrococcus spp. (9.4 %) were the most common halophilic gram-positive bacteria. All Bacillus species discovered started the fermentation, however only B. subtilis, B. licheniformis and B. megaterium survived until the end of the fermentation process. Xiao, et al. (Xiao, Zhao, Wu, Lin, Zhang, & Gao, 2014) isolated fifty-five protease-producing bacteria from ten fermented fish sauce samples and identified them using the 16S rDNA gene sequences. BLAST examination of the 16S rDNA sequences revealed that forty-six strains belonged to Bacillus spp. Ngari is the most popular fermented fish product in Northeast India, made exclusively from sun-dried tiny carp Puntius sophore Ham, also known locally as phabou. It is generated by natural fermentation for up to a year and is used as an aperitif and flavor enhancer in a variety of culinary dishes due to its exceptional organoleptic features.

A total of 210 bacteria isolated from the samples were identified using ARDRA-based grouping and 16S rRNA gene sequence similarity analysis. The dominating bacteria were Staphylococcus cohnii (38.0 %), Tetragenococcus halophilus (16.8 %), a new phylotype linked to Lactobacillus pobuzihii (7.2 %), Enterococcus faecium (7.2 %), B. indicus (6.3 %), and Staphylococcus carnosus (3.8 %) (Devi, Deka, & Jeyaram, 2015). Kapi, a traditional fermented shrimp paste from Thailand, is commonly used as a condiment. Small shrimp (Acetes vulgaris) and krill (Mesopodopsis orientalis) are commonly used as components, along with sea salt in a 5:1 ratio. After drying and grinding, the mixture is stored in jars at room temperature for many months, or until the characteristic perfume emerges. Traditional fermented foods are deemed healthy due to their long history of consumption, but they have not been examined for hygienic and microbiological safety. When their microbiota was examined using 16S rRNA gene amplicon sequencing, Bacillus spp. were discovered (Nakamura et al., 2022, Yiamsombut et al., 2021).

Commercially available probiotic Bacillus spore products

Bacillus strains are widely distributed in the natural environment, including soil, air, fermented foods, and the human intestinal tract. Probiotic Bacillus spores are resistant to harsh environmental conditions, which allows them to survive in situations when other vegetative bacteria could die off (Gao et al., 2023). The ability of probiotic Bacillus spores to germinate, grow, and regenerate in the gastrointestinal tract has been demonstrated (Fig. 2). Spores of probiotic Bacillus spp. have been demonstrated to momentarily coexist as commensal organisms within the host, and it is thought that the spores of these species germinate or persist in the small intestine, where they regulate intestinal conditions. Many items containing Bacillus spores are sold as “novel foods” or dietary supplements with the promise of improving user health and reestablishing the intestinal tract's natural microbiota (Zhao et al., 2023). Bacillus spore products are primarily used as growth promoters and feed additives in animal and aquaculture farming (Ramlucken et al., 2020). This review primarily focuses on human applications of spore products formed by probiotic Bacillus, providing a brief summary of the major Bacillus spp. used in commercial products (Table 2). Most commercial Bacillus probiotics include B. coagulans, B. subtilis, B. polyfermenticus, B. clausii, B. cereus, B. pumilus, and B. licheniformis. Interestingly, the fact that many Bacillus spore products are licensed as human pharmaceutical supplements confirms that these B. coagulans are safe for consumption.

Fig. 2.

Schematic diagram of the forms of probiotic bacillus used and their functions on the human body.

Table 2.

Probiotic Bacillus spores in commercial products.

Product	Manufacturer	Comments	References
Primal Defense	Garden of life, USA	Combination of 12 probiotics, one of which includes B. subtilis DE-111	(Gallart, Sanseverino, & Winger, 2020)
MegaSporeBiotic	Microbiome Labs,USA	B.indicus, B. subtilis, B. coagulans, B. licheniformis, B. clausii	(Elshaghabee et al., 2017)
Biosubtyl	Biophar Company, Nha Trang, Vietnam	Sachet (1 g) carrying 106 –107 of B. pumilus spores mixed with tapioca. Product labelled as B. subtilis	(Duc et al., 2004, Soares et al., 2023)
Bispan	Binex Co. Ltd, Busan, Korea	Tablet carrying spores (1.7 × 107) of B. polyfermenticus SCDd	(Paik, Park, & Park, 2005)
Domuvar	BioProgress SpA, Anagni, Italy	Vial carrying 1 × 109 spores of B. clausii in suspension, labelled as carrying B. subtilis	(Cutting, 2011, Lee et al., 2019)
Flora-Balance	Flora-Balance, Montana, USA	Capsules labelled as carrying Bacillus laterosporus BODd but containing Brevobacillus laterosporus BOD	(Sanders, Morelli, & Tompkins, 2003)
Lactospore	Sabinsa Corp., Piscataway, NJ, USA	Labelled as carrying Lactobacillus sporogenesd but contains B. coagulans 6–15 × 109 g−1	(David, Katy, & Judith, 2010)
Medilac	Hanmi Pharmaceutical Co. Ltd., Beijing, China	B. subtilis strain RO179 (at 108 g 1) in combination with Enterococcus faecium	(Hong, Duc, & Cutting, 2005)
Natur’s First Food	Nature s First Law, San Diego, CA, USA	42 species listed as probiotics including: B. subtilis, B.polymyxa B.pumilus and B. laterosporus	(Sanders et al., 2003)
Neolactoflorene	Newpharma S.r.L., Milan, Italy	L. acidophilus, B. bifidum and L.sporogenes. L.sporogenes at 3.3 × 105 CFU/g whose valid name is B. coagulans is mislabelled and is a strain of B. subtilis	(Fasoli et al., 2003)
Subtyl	Mekophar, Pharmaceutical Factory No. 24, Ho Chi Minh City, Vietnam	Capsule carrying 106 –107 spores of a B. cereus species termed B. cereus var vietnami. Product labelled as carrying B. subtilis	(Soares et al., 2023)

Beneficial metabolites

Enzyme-rich resources

Food fermentation is a biochemical process based on the metabolism and action of microorganisms, in which certain components are transformed into other compounds, thus changing the form, taste, and aroma of the food product. Bacillus spp. has the ability to produce high amounts of protease, lipase, and amylase, which play a vital role in food fermentation, improving not only the quality and taste of food, but also the nutritional value.

Currently, Bacillus species represent the primary bacterial source of proteases, exhibiting the ability to generate elevated quantities of both alkaline and neutral proteolytic enzymes, each with unique characteristics. Proteases produced by Bacillus and their applications in food are summarized in Table 3. Sanjukta et al. (Sanjukta, Rai, Muhammed, Jeyaram, & Talukdar, 2015) hydrolyzed soy protein using B. subtilis MTCC5480 and B. subtilis MTCC1747 proteases, and the released peptides and free amino acids presented strong antioxidant properties. Zhao et al. (Zhao et al., 2012) showed that the cold-adapted collagenolytic protease MCP-01 secreted by B. subtilis BEM01 had a stronger tenderizing effect on meat samples than papain, and meat tenderization at low temperatures was more conducive to maintaining the freshness and quality of meat. Sorapukdee et al. (Sorapukdee, Sumpavapol, Benjakul, & Tangwatcharin, 2020) found that the proteases secreted by B. subtilis B13 and B. siamensis S6 efficiently hydrolyzed bovine Achilles tendon collagen and that these two Bacillus proteases had higher hydrolytic activity on elastin and beef intramyocardial collagen and lower hydrolytic activity of beef myofibrillar proteins than papain and pineapple protease. B. licheniformis LBA46 protease can be used to hydrolyze pea protein to prepare pea protein peptides. The hydrolysis conditions of pea protein were optimized using response surface methods, and the best conditions were determined to be a pH of 10 and an enzyme concentration of 100 U/mL. The hydrolysis product has a strong 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging ability, oxygen radical absorbance, and ferric ion reducing/antioxidant power, indicating some application potential (Aguilar & d. S., Castro, R. J. S. d., & Sato, H. H. , 2020). Yang et al. (Yang et al., 2016) found that the extracellular proteases secreted by B. licheniformis CGMCC 0635 were able to improve the nutritional properties of peanut proteolytic digests, such as the levels of crude protein, organic acids, acid-soluble oligopeptides, and minerals, and to enhance the antioxidant properties, amino acid balance, and in vitro digestibility. Thermolysin, secreted by B. thermophilus, digested rice bran and produced peptides with antihypertensive properties (Shobako et al., 2018). Song et al. (Song et al., 2021) heterologously expressed and purified B. cereus CMCC 63303 collagenase and applied it to the hydrolysis of bovine bone collagen to prepare collagen peptides.

Table 3.

Beneficial metabolites produced by probiotic Bacillus.

Product type	Probiotics Bacillus speices	Beneficial metabolite	Effectiveness	References
Enzymes	B. subtilis MTCC5480 B. subtilis MTCC1747	Fibrinolytic enzyme and proteolytic enzyme	Soybean fermented with possible proteolytic B.subtilis strains had higher antioxidant capabilities due to increased peptides and polyphenols.	(Sanjukta et al., 2015)
	B. subtilis BEM01	Cold-adapted collagenolytic protease MCP-01	MCP-01 significantly reduces beef shear when applied at 4 °C, preserving meat's bright color and moisture. Unlike commercially available papain and bromelain, MCP-01 is highly selective for collagen degradation at 4 °C.	(Zhao et al., 2012)
	B. subtilis B13 B. siamemsis S6	Serine proteases and some metalloproteases	In contrast to papain and bromelain, these collagenolytic proteases showed strong hydrolysis toward collagen and elastin as well as beef intramuscular collagen with low beef myofibrillar protein degradation.	(Sorapukdee et al., 2020)
	B. subtilis	B. subtilis protease powder (CTC E-ssentials MT-70 N)	B. subtilis protease can be used as a meat tenderizer instead of current commercial tenderizers that contain plant-derived proteases.	(Bureros, Dizon, Israel, Abanto, & Tambalo, 2020)
	B. licheniformis LBA46	Alkaline protease LBA	B.licheniformis pea protein hydrolysate exhibits significant DPPH radical rate, oxygen radical uptake and iron ion reducing antioxidant capacity.	(Aguilar & d. S., Castro, R. J. S. d., & Sato, H. H. , 2020)
	B. licheni formis CGMCC0635	Extracellular	It improves the crude protein, organic acids, acid-soluble oligopeptides and other actives, minerals and antioxidant properties, amino acid balance and in vitro digestibility in peanut protein, and enhances the nutritional and functional properties of peanuts.	(Yang et al., 2016)
	B. thermophilus	Thermolysin	The antihypertensive activity of protease-digested rice bran in a spontaneously hypertensive rat (SHR) paradigm.	(Shobako et al., 2018)
	B. cereus CMCC63303	Collagen enzyme	Under ideal conditions (110.0 μg/mL collagenase concentration, 35 °C, pH 8.0, and 6.0 h), collagenase demonstrated hydrolytic activity.	(Song et al., 2021)
	B. subtilis WB800	Geobacillus stearothermophilus lipase (T1.2RQ)	Excellent hydrolytic and transesterification activity.	(Elemosho et al., 2021)
	B.subtilis KM-BS	Lipase EstA	EstA, a recombinant lipase, demonstrated strong activity (49.67 U/mL), as well as high heat and pH stability.	(Nguyen et al., 2024)
	B. pumilus V1, B. pumilus V7, and B. subtilis V8	Extracellular lipase	The bacterial cultures B. pumilus V1, B. pumilus V7, and B. subtilis V8 produced 27.8, 25.2, and 16.6 µg/mL of lipase respectively.	(Kandasamy et al., 2021)
	B.subtilis strain US586	Acid-stable alpha-amylases	Adding pure amylolytic extract from the newly identified B. subtilis strain US586 to weak local flour enhances dough rheological properties and bread quality.	(Trabelsi et al., 2019)
	B. amyloliquefaciens BH072	α-amylase	The reported α-amylase enzyme has a specific activity of 2162.42 U/mg and a molecular mass of approximately 68 kDa.	(Du et al., 2018)
Antimicrobial substances	B. subtilis SC3.7	Subtilosin A	The strongest zone of inhibition was reported against K. rhizophila, followed by L. monocytogenes and other bacterial species.	(Epparti et al., 2022)
	B. subtilis R0179	Iturin A	It significantly inhibits the growth of Candida species.	(Zhao, Wang, et al. 2021)
	B. subtilis N7	Mejucin（a novel bacteriocin）	Mejucin have bactericidal effects on both vegetative and spore cells of B. cereus.	(Lee & Chang, 2018)
	B. subtilis NT-6	Antimicrobial peptide AMPNT-6	AMPNT-6 proved effective in controlling biofilms on various contact surfaces by lowering adhesion and/or eliminating biofilm.	(Deng et al., 2017)
	B. subtilis	Amicoumacin A	It has been shown to be antagonistic against Enterobacteriaceae species, as well as to inhibit Helicobacter pylori.	(Zhao et al., 2016)
	B. natto	Surfactinv	It may inhibit the growth of Candida albicans in the digestive trac.	(Jakab et al., 2022)
	B. megaterium	A bacitracin like peptide compound	It has exhibited potential broad spectrum antimicrobial activity.	(Abdullah et al., 2018)
	B. clausii	Gallidermin	Gallidermin suppresses staphylococci growth in a dose-dependent manner while also effectively preventing biofilm formation by Staphylococcus aureus and Staphylococcus epidermidis.	(Saising et al., 2012)
	B. licheniformis ATCC 14580	Lichenicidins	Lichenicidin is a two-peptide lantibiotic with antibacterial activity against all Listeria monocytogenes, methicillin-resistant Staphylococcus aureus, and vancomycin-resistant Enterococcus strains tested.	(Begley et al., 2009)
	Clostridium beijerinckii ATCC25752	Cirucularin A	Clostridium beijerinckii ATCC25752 produces Circularin A, which is active against Clostridium tyrobutyricum, a recognized cheese-spoilage bacterium.	(Kawai et al., 2004)

Lipases are very essential commercial enzymes that catalyze the hydrolysis of triglycerides to glycerol and free fatty acids, laying the groundwork for the production of taste compounds during food fermentation. Elemosho et al. (Elemosho, Antonius, & Maggy, 2021) reported successful extracellular expression of thermophilic lipase (T1.2RQ), a new industrially desirable heat-stable lipolytic enzyme with outstanding hydrolytic and ester-exchange activity, in B. subtilis WB800. Disha Sharma et al. optimized the key growth parameters to enhance the extracellular lipase yield produced by B. alkalophilus KS4 3.54-fold. Nguyen et al. (Nguyen et al., 2024) cloned the lipase gene estA from B. subtilis KM-BS and optimized it for effective heterologous protein production in Escherichia coli BL21 (DE3) cells. The ability of this pure recombinant lipase to hydrolyze waste cooking oil was biochemically characterized.

Amylases are extracellular enzymes that randomly hydrolyze starch molecules to yield a variety of products, including dextrins and progressively smaller polymers made up of glucose units. Trabelsi et al. (Trabelsi, Mabrouk, Kriaa, Ameri, Sahnoun, Mezghani, & Bejar, 2019) found that Amy586 secreted by B. subtilis strain US586 improves bread textural parameters by decreasing bread hardness and increasing cohesion and elasticity values. Du et al. (Du et al., 2018) isolated and characterized a new α-amylase generated by B. amyloliquefaciens BH072, which showed high hydrolysis rates of maize, wheat, and potato starch and hydrolyzed soluble starch to glucose, maltose, maltotriose, and maltotetraose.

Antibacterial substances

Bacteriocins are proteins generated by bacteria that serve as antimicrobial agents, primarily inhibiting the growth of closely related bacteria. Bacteriocin production is a good trait of probiotic strains, which are inherently non-toxic and have excellent bacteriostatic action against food spoilage bacteria and human infections. A number of Bacillus species produce bacteriocins which have a significant inhibitory effect on pathogenic or endogenous infections by conditionally pathogenic bacteria, some notable examples of which are listed in Table 3.

B. subtilis is thought to be one of the most efficient “antibiotic-producing” species in the Bacillus genus, with antibiotic production accounting for 4 %-5% of its genome. In total, 66 antibiotics have been identified in different strains of B. subtilis, suggesting that this species has important biotechnological potential. For example, Subtilosin A produced by B. subtilis had the strongest inhibitory effect on Rhizopus, followed by Listeria monocytogenes and other bacterial strains (Epparti, Eligar, Sattur, & Halami, 2022). Iturin A produced by B. subtilis R0179 has a significant inhibitory effect on the growth of Candida (Zhao, Lou, Shui, Zhang, Hu, Zhang, Li, Wu, & Li, 2021). Mejucin, a novel bacteriocin, has a bactericidal effect on the trophoblasts and sporoblasts of B. cereus (Lee & Chang, 2018). Amicoumacin A generated by B. subtilis is an antibiotic with anti-inflammatory effects that can be used to treat Helicobacter pylori infection (Zhao et al., 2016). B. natto produces surfactin, which may prevent the growth of Candida albicans in the digestive system (Jakab et al., 2022). Similarly, Abdullah et al. (Abdullah et al., 2018) investigated the production of a broad-spectrum lipopeptide (a bacitracin-like peptide compound) from B. megaterium, which is highly effective against both Gram-positive and Gram-negative bacterial pathogens. Saising et al. (Saising et al., 2012) demonstrated that Gallidermin suppressed not only the growth of Staphylococcus in a dose-dependent manner but also effectively prevented biofilm formation. B. licheniformis ATCC 14580 developed a dipeptide antibiotic called Lichenicidin, which was effective against L. monocytogenes, methicillin-resistant Staphylococcus aureus, and vancomycin-resistant E. faecium (Begley, Cotter, Hill, & Ross, 2009). Clostridium beijerinckii ATCC 25752 produces Circularin A, which is active against Clostridium tyrobutyricum, a bacteria that causes cheese deterioration. (Kawai, Kemperman, Kok, & Saito, 2004).

Probiotic Bacillus as fermentation agents

With the in-depth and extensive research on Bacillus probioticus, it is slowly being used as fermentation agent in single fermentation or co-fermentation with Lactobacillus, and the latest results are shown in Table 4. The probiotic Bacillus spp. used for food fermentation include B. subtilis, B. coagulans, B. velezensis, B. amyloliquefaciens, B. pseudomycoides, B. natto, B. licheniformis, B. brevis, and B. cereus. It is exciting that the use of probiotic Bacillus strains in fermented foods imparts a richer flavor and texture to the products, increases the nutritional value of the products, improves the safety of the products and degradation rate of the products to a certain extent, shortens the fermentation time, and provides a new way to reduce salt levels in fermented foods. Of these, B. coagulans has been reported to be safe by the US Food and Drug Administration and the European Union Food Safety Authority, and it is on the GRAS and QPS lists. In addition, genome sequencing has been reported to provide information about the overall characteristics of the bacteria, which can better demonstrate its safety as a food supplement. The genome of B. coagulans GBI-30 was sequenced and was found to be free of any deleterious genes.

Table 4.

Probiotic Bacillus as food fermentation agents.

Species	Fermentation mode	Food	Contribution	References
B. subtilis WX-17	Single fermentation	Probiotic beverage	Essential amino acids and short-chain fatty acids were dramatically increased. Total phenolic content and antioxidant content (measured by DPPH radical scavenging activity) rose by 6.32 and 1.55, respectively.	(Mok, Tan, Lyu, & Chen, 2020)
B.subtilis LK-1	Single fermentation	Dark tea	New flavor substances such as geranyl isovalerate are produced after fermentation, and the floral and fruit aroma of dark tea improved with fermentation.	(Xiao et al., 2023)
B. subtilis lwo	Single fermentation	Chickpeas	The proteolysis and the antioxidative properties of chickpeas were improved by solid-state fermentation.	(Li & Wang, 2021)
B. subtilis Y61	Single fermentation	Sichuan paocai	The inoculated fermentation of B. subtilis Y61 can improve the quality and safety of SCP while also shortening fermentation time.	(Yang et al., 2020)
B. coagulans	Single fermentation	Tender Coconut Water	The synthesis of exopolysaccharides by B. coagulans during fermentation resulted in a considerable increase in the soluble solids content and viscosity of the fermented coconut water.	(Gangwar, Bhardwaj, & Sharma, 2018)
B. coagulans VHProbi C08	Single fermentation	Plant-based drink	Plant-based drink fermented by VHProbi C08 has great antibacterial and antioxidant activities.	(Shudong et al., 2022)
B.velezensis CS1.10S	Single fermentation	Soy sauce	CS1.10S is salt tolerant and improves the taste and aroma of soy sauce.	(Bai et al., 2023)
B. clausii	Single fermentation	Spent coffee grounds (SCG)	Peptides with strong bioactive potential are more abundant in fermented SCG and can be used to treat diabetes, hypertension, and oxidative stress.	(Ramírez, Pineda-Hidalgo, & Rochín-Medina, 2021)
B. natto	Single fermentation	Peanuts	The method reduced more than 77.3 % of the IgE reactivity in peanut protein preparations.	(Pi et al., 2021)
B. natto	Single fermentation	Rosa roxburghii pomace	B. natto helps to produce high-quality dietary fiber from Rosa roxburghii pomac.	(Chu et al., 2019)
B. coagulans MTCC 5856 and Streptococcus thermophilus	Cooperative fermentation	Fermented dairy	B. coagulans coupled with S. thermophilus produce a dairy product of improved quality, and the product has a high antioxidant activity enhancing its therapeutic value.	(Lavrentev et al., 2021)
Enterococcus, Lactobacillus, Bacillus, and Leuconostoc	Cooperative fermentation	Amaranth doughs	Fermentation of amaranth with LAB and Bacillus spp. allowed the release of protein hydrolysates with antioxidant, antihypertensive, and antimicrobial activity.	(Cruz-Casas et al., 2023)
B. amyloliquefaciens MK063714 and Candida versatilis MK063708	Cooperativefermentation	Horse bean	Combination of B. amyloliquefaciens and C. versatilis could obtain more extensive aroma profiles, especially for the enrichment of miso-like and fruity flavors.	(Lu, Yang, Yang, Chi, & He, 2021)
B.amyloliquefaciens HZ-12,Escherichia coli DH5α	Cooperativefermentation	Soybean	The lycopene yield of B. amyloliquefaciens HZ-12/pHYcrtEIB was further increased.	(Zou et al., 2022)
Saccharomyces cerevisiae and B.licheniformis	Cooperative fermentation	Jujube wine	Co-fermentation enhances the flavor of jujube wine, as evidenced by substantial differences in color, taste, amino acids, organic acids, and aroma (p < 0.05). Co-fermented jujube wine also has superior color quality.	(Zhao et al., 2022)
B. subtilis and B. coagulans	Cooperative fermentation	Soybean residue	B. subtilis R0179 and B. coagulans 123 reduced undesirable green and beany flavours in okara.	(Keong, Toh, Lu, & Liu, 2023)
Aspergillus oryzae and B.subtilis	Cooperative fermentation	Gochujang meju	Acid protease has high activity and free amino acid content.	(Kim, Han, & Kim, 2010)
B.cereus DM423 and L.plantarum HH-LP56	Cooperative fermentation	Pork	B. cereus DM423 can promote flavor formation in fermented sausages	(Shan et al., 2023)

A large number of Probiotic Bacillus strains have been isolated from traditional natural fermented foods. Probiotic Bacillus strains have long been used by consumers in the form of fermented foods, but it has been neglected and its role in human health and food fermentation has not been emphasized. Bacillus is an understudied bacterial genus, which is commonly found in many fermented foods, especially solid-state fermented foods. Certain probiotic Bacillus species generate large amounts of lipases and proteases, as well as extracellular polymeric compounds and strong antifungal lipopeptides that are uncommon in other significant food fermenters, such as Lactobacillus, Acetobacter, or Propionibacterium. Further research into the probiotic properties of Bacillus spp. will support their development into next-generation fermentation agents for the production of many new and improved fermented foods.

Opportunities and challenges

Opportunities

As consumer habits evolve and interest in food and health grows, there is an increasing demand for healthier and more practical food options. Traditional fermented foods, primarily produced at home or in small-scale settings, face the challenge of modernized manufacturing techniques in traditional food enterprises. The integration of ancient traditional fermentation processes with modern technologies poses new challenges and opportunities for the food industry. In particular, the exploration and enrichment of microbial resources, coupled with the development of diverse microbial fermentation agents, are crucial tasks. Probiotic Bacillus strains, with their potential to become next-generation probiotic fermentation agents, play a key role in meeting this demand.

Many probiotic Bacillus strains can be isolated from traditional naturally fermented foods, especially solid-state fermented products. However, research on their role as fermenting agents in food fermentation is limited and not comprehensive. Some probiotic Bacillus strains possess excellent probiotic characteristics that remain underexplored. Once characterized, these strains can be used for co-fermentation with the now common probiotics (L. plantarum, L. lactis) to make food products with a better flavor and higher nutritional value. Simultaneously, the spores produced by Bacillus probioticus provide a new nutritional enhancer for heat-treated probiotic products due to their heat resistance, which can be of interest to food companies and consumers. The longstanding presence of probiotic Bacillus strains in traditional fermented foods, coupled with existing reports on their probiotic properties and fermentation performance, paves the way for further comprehensive research and exploration of their potential as fermentation agents.

Furthermore, with the advancement of scientific and technological capabilities, the refinement of omics technologies such as genomics, proteomics, and metabolomics has allowed us to gain a deeper understanding of organisms. This enhanced understanding includes the fundamental composition and evolutionary patterns of living organisms. Through genomic analysis at the gene level, we can predict and analyze functional genes, gene clusters related to the synthesis of bioactive metabolites, resistance genes, and independent factors. These advancements in omics technologies propel the research and development of probiotic Bacillus strains, enabling a more comprehensive exploration of their capabilities and potential.

Challenges

The presence of probiotic Bacillus spp. in most traditional natural fermented foods confirms that probiotic Bacillus strains have a long application history. However, several Bacillus spp. provide dangers, including the formation of toxic metabolites and toxins, the transmission of antibiotic resistance genes, the generation of excess amines, and infection. Therefore, the use of probiotic Bacillus strains as food fermentation agents must be adequately investigated with more detailed phenotypic and genotypic safety assessments to ensure that it is safe.

Probiotic Bacillus strains, being novel fermentation agents in the development of new fermented foods, introduce new and unknown components, leading to public caution. Thermophilic Bacillus spp. and their metabolites represent a new category of food components. To promote consumer health, operators must provide extensive information on the new food product prior to manufacture and sale, while complying to regulations and processes unique to each country and region. The procedures for approving the market entry of new foods vary by location, and there is a lack of globally unified regulatory standards for the assessment and oversight of new foods. In order to address this, it is crucial for regulatory bodies worldwide to establish consistent standards for the evaluation and regulation of new foods to ensure consumer safety.

Conclusion

This article provides an overview of the presence of probiotic Bacillus strains in the microbial composition of liquid, semi-solid and solid fermented foods, and probiotic Bacillus strains widely exist in solid fermented foods. Furthermore, the statistics of probiotic Bacillus products currently on the market, and the production of enzymes and antibiotics by probiotic Bacillus strains are conducted to illustrate their edible safety and fermentation feasibility. Finally, a statistical analysis is performed on the research on single fermentation and mixed fermentation of probiotic Bacillus strains as food fermentation agents. Excitingly, the use of probiotic Bacillus strains in food fermentation enhances the flavor and texture of the product, increases the nutritional value, and improves the safety. Additionally, it contributes to the metabolism of raw food material, shortens fermentation times, provides a new avenue for salt reduction in fermented foods, and offers novel approaches for modernizing manufacturing techniques in traditional food enterprises. The integration of modern and ancient traditional fermentation processes has been made possible through the utilization of probiotic Bacillus strains, paving the way for innovative developments in the fermented food industry.

Based on this, we can focus on the following research directions in the future: (1) Further explore the resources of probiotic microorganisms in traditional natural fermented foods, and use more accurate microbial diversity characterization technology (such as the third generation sequencing technology, etc) to make the safety and probiotic properties of probiotic Bacillus strains more clear. (2) Combined use of multi-omics technologies such as genomics, proteomics and metabolomics to further explore the pathways of probiotic Bacillus on microbial composition and complex flora material metabolism in the traditional natural fermented food process, and clarify the positive contribution of probiotic bacteria in the food fermentation process. (3) Explore the flavor substance formation mechanism of probiotic Bacillus collaborative fermentation in traditional fermented foods, and achieve intelligent and precise control of the brewing process of traditional fermented foods to produce better-quality and more delicious foods.

CRediT authorship contribution statement

Shijie Liu: Writing – review & editing, Writing – original draft, Investigation. Lijun Zhao: Writing – review & editing, Formal analysis. Miaoyun Li: Writing – review & editing, Supervision, Resources, Funding acquisition. Yaodi Zhu: Writing – review & editing. Dong Liang: Supervision. Yangyang Ma: Supervision. LingXia Sun: Supervision. Gaiming Zhao: Supervision. Qiancheng Tu: Supervision.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability

Data will be made available on request.