Here, we characterized the microbiotas of Thua Nao from different areas of northern Thailand and identified the beneficial bacteria potentially synthesizing SCFAs. We believed that the raw materials used in Thua Nao production (i.e., soybean, and the natural flora of each area) would yield diverse bacterial classification results. We also assessed two types of Thua Nao, wet and dried, using 16S rRNA gene sequencing, a culture-independent method. At the phylum level, Firmicutes was the most abundant and common in both wet and dried soybean samples. Members of Firmicutes mainly include beneficial bacteria that play significant roles in the relationship between gut bacteria and human health. Some Firmicutes members can ferment dietary fiber from legumes and produce metabolites, including SCFAs 7. We further analyzed the predominant genera from Firmicutes with ≥ 1% abundances. At the genus level, Bacillus was the most abundant in both wet and dried samples, which was consistent with previously reported microbial compositions 2,3,8.
Considering the differences in bacterial compositions between wet and dried Thua Nao, differential abundance analyses showed high abundances of lactic acid bacteria (LAB). LAB is used to produce various animal and vegetable products and for developing flavors, aromas, and textures in fermented foods 9. The different proportions of LAB in the wet and dried samples could be factors in the viability of LAB in preserved Thua Nao. Our results showed that two LAB genera, Lactobacillus and Enterococcus, were highly abundant in the wet samples. Lactobacillus strains can hydrolyze soy proteins 10. Lactobacillus spp. are commonly used as probiotics for human and animal supplements, and mixed cultures of Lactobacillus are used to ferment soybean meal 11. The wet samples also contained high abundances of enterococci. Although some enterococci are considered pathogenic determinants, these bacteria also have some positive features. Some enterococci have been described as potential starters in various fermented products. The use of enterococci as starter cultures or co-cultures has increased considerably. The mixed natural flora of predominant Bacillus, LAB, and other bacteria might create unique flavors, aromas, and textures in wet Thua Nao.
Thua Nao is a popular fermented food in northern Thailand (i.e., Chiang Mai, Chiang Rai, Lampang, Lamphun, Mae Hong Son, and Phayao) because it can be used as an ingredient to increase the flavor in cooked foods and can be eaten directly with sticky rice or rice 1. Various factors in Thua Nao manufacturing, including the types and amount of fibers in raw soybeans, the natural microbiota, environment, and fermentation duration vary among the provinces, resulting in distinct microbiome profiles and different tastes of Thua Nao. Our results indicate that Thua Nao samples from Lampang, Chiang Mai, and Chiang Rai had higher microbial diversity than those from Lamphun, Mae Hong Son, and Phayao in the wet condition.
Thua Nao contains high concentrations of non-digestible carbohydrates, which cannot be digested by endogenous enzymes in the stomach and small intestines, but are a potential fermentation substrate for beneficial microbes in the gut and are considered prebiotics 12–14. Fermentation using natural beneficial microbes in fermented food production involves hydrolyzing non-digestible carbohydrates to sugars, which are subsequently fermented to produce primarily SCFAs as well as H2 and CO2 14,15. Several studies reported that the types and amounts of SCFAs depend greatly on bacterial digestibility and the fermentability and viscosity of fiber 14,15. The three main SCFAs, acetic acid (C2), propionic acid (C3), and butyric acid (C4), provide energy sources for gut microbial growth and physiological effects in humans 14. These SCFAs are carbon-volatile fatty acids and non-stable. The most abundant of these SCFAs is produced by anaerobic fermentation of dietary fibers 5,16. These probiotic metabolites were significantly higher in wet Thua Nao than in dried Thua Nao, possibly owing to the characteristics of volatile SCFAs and destroyed anaerobic enzymatic activities during drying 13,16,17.
Acetic acid was the most abundant SCFA in both the wet and dried Thua Nao samples. Clostridium, Ignatzschineria and LAB, which are anaerobes, and acetic acid bacteria (AAB), which are aerobes, are mainly responsible for acetic acid production 18–20. Therefore, Clostridium species (i.e., Clostridium sensu stricto), Ignatzschineria, LAB (i.e., Lactobacillus, Pediococcus, Streptococcus, Enterococcus, Globicatella, Vagococcus, and Weissella) and AAB (i.e., Acetobacter) in Thua Nao might be associated with acetic acid production during fermentation. Acetic acid concentrations were the most abundant in Chiang Rai and Lampang, corresponding to the highest amounts of LAB, AAB, Ignatzschineria, and Clostridium spp.
Propionic acid bacteria produce propionate, a primary fermentation product 20,21. The three major propionate formation pathways are the succinate, acrylate, and propanediol pathways 22. Propionibacterium and Clostridium, which are anaerobes, have been explored as potential propionate producers via the succinate pathway 18,23. Correlation analysis showed that the propionate concentrations were associated with Globicatella, Ignatzschineria, Clostridium sensu stricto, Paenalcaligenes, and Staphylococcus abundances. Clostridium, Ignatzschineria, and Paenalcaligenes are reported to be potential propionic acid producers 24,25. Although Ignatzschineria and Paenalcaligenes belong to the phylum Proteobacteria, implicated in human infections 26, these genera were found in very low amounts (< 1%) in dried Thua Nao. The highest amount of propionic acid was observed in Lampang, followed by Chiang Mai, and corresponded to the highest relative abundances of Globicatella, Clostridium sensu stricto, and Staphylococcus.
Correlation analysis showed that the butyrate concentration was associated with the abundances of Globicatella, Ignatzschineria, Corynebacterium, Sphingobacterium, Clostridium sensu stricto, Vagococcus, Paenalcaligenes, Staphylococcus, and Comamonas. Previous studies showed that butyrate can be generated from the fermentation of LAB such as Globicatella, Vagococcus, Staphylococcus; Bacillus spp.; Corynebacterium, Comamonas, Sphingobacterium, Ignatzschineria; and Clostridium sensu stricto 27–29. The highest amounts of butyric acid were observed in Chiang Mai, followed by Lampang, and corresponded to the highest relative abundances of Corynebacterium in Chiang Mai, Sphingobacterium and Vagococcus in Chiang Mai and Lampang, Globicatella and Clostridium sensu stricto in Lampang, and Comamonas in Mae Hong Son. Correlation analysis showed that the total SCFA concentrations were associated with the relative abundances of Weissella and Lactobacillus. The highest amounts of total SCFAs observed in Chiang Rai corresponded to the highest relative abundances of Weissella and Lactobacillus.
Our results indicate that both wet and dried Thua Nao from the northern provinces of Thailand are potential sources of probiotics and SCFAs. Notably, we used an amplicon-based microbiome approach to observe the bacterial communities in Thua Nao, and the results yielded only genus-level bacteria. This study provides insight into SCFA-microbiota associations in Thua Nao, which could be used to develop starting cultures for SCFA-enriched fermented soybean production as a supplementary food to modulate lifelong health. The effect of the microbiome in Thua Nao and metabolite production (e.g., SCFAs) should be further investigated using other approaches, including shotgun metagenomics, genome sequencing, and metabolomics.