Application of Metabonomics in the Research of Lactic Acid Bacteria-fermented Foods

Guangpeng LIU Yanrui MA Le CHU Fengtao ZHU Yongbo DOU Jiwu WANG Fatao HE

Abstract Lactic acid bacteria have been important industrial floras in the food fermentation production industry since ancient times. Their metabonomics technology has been widely used in the process monitoring, product classification and flavor formation mechanism of lactic acid bacteria-fermented foods. In this study, we discussed the application status of metabonomics technology in lactic acid bacteria-fermented food industry and scientific research, including dairy products, soy products, beverages and some pickled foods. Metabolomics will be applied in the research fields of lactic acid bacteria metabolism mechanism of lactic acid bacteria-fermented foods, lactic acid bacteria metabolomics database construction and expansion, metabolomics and genomics, and proteomics. In particular, the metabolomics technology will be introduced into product development and quality monitoring of traditional Chinese lactic acid bacteria-fermented foods, such as fermented bean curd and suancai, thereby perfecting industry norms and improving the level of industry development.

Key words Metabolomics; Lactic acid bacteria; Fermentation; Food

Received: February 14, 2021  Accepted: April 17, 2021

Supported by Yantai Science and Technology Planing Project (2019ZDCX013); High-efficiency Ecological Agriculture Innovation Project of Taishan Industry Leading Talent Engineering (LJNY202001); Key R&D Program of Shandong Province (2019GNC106019).

Guangpeng LIU (1984-), male, P. R. China, researcher, devoted to research about fruit and vegetable fermentation.

*Corresponding author. E-mail: liuguangpeng99@163.com.

Lactic acid bacteria have been involved in many fermented food production processes as common strains in fermented foods since ancient times, such as dairy products［1］, soy products［2］, drinks［3-4］ and some pickled foods［5］. The metabolic activities of lactic acid bacteria are closely related to the flavor, aroma, texture and nutrition of fermented foods. With the development of metabonomics technology, this technology has been widely used in the research of monitoring the process of lactic acid bacteria-fermented foods and the formation mechanism of characteristic flavor substances.

Lactic acid bacteria will produce a large number of metabolites during fermentation, including organic acids, amino acids, oligosaccharides, small peptides and some aromatic substances, etc., and revealing the relationship between these metabolites and their influencing factors is of great significance for improving the quality of fermented foods based on lactic acid bacteria and optimizing the fermentation process. The comprehensiveness and high resolution of metabolomics can comprehensively evaluate and discover the subtle differences between samples, so it can provide conclusive and quantitative data for lactic acid bacteria-fermented foods, fermentation process optimization and product quality improvement.

Application of Metabonomics in Fermented Dairy Products

Lactic acid bacteria are widely used in fermented dairy products, including yogurt, cheese and kefir, which are dairy products mainly fermented with lactic acid bacteria. Among fermented dairy products, Lactococcus, Lactobacillus, Pediococcus, Enterococcus and Leuconostoccus are commonly used strains of fermented milk products. They metabolize the esters, sugars, and proteins in milk, and add unique taste and flavor to fermented dairy products through small molecular compounds from the gradually degradation of above substances. In recent years, in order to more finely control the fermentation process of lactic acid bacteria, metabonomics technology has been widely used in the research of fermented dairy products, aiming to explore the relationship between lactic acid bacteria and fermented milk quality structure, taste and nutrition. Mazzei and Piccolo used NMR technology to study the flavor and nutritional characteristics of the famous mozzarella cheese with special flavor. The study found that mozzarella cheese produced more isobutanol, lactic acid and acetic acid during the fermentation process compared with other cheeses, and the mozzarella cheese produced in this area could be clearly identified through metabolomics research［6］. Piras et al.［7］ applied metabolomics to study the role of lactic acid bacteria in the maturation process of Italian traditional Fiore Sardo cheese, and found that lactic acid bacteria floras formed by natural fermentation had obvious differences in flavor compared with commercial lactic acid bacteria starters, and commercialized lactic acid bacteria starters could not produce the designated protection of origin products certified by the EU agricultural product inspection agency. Ochi et al.［8］ used GC-TOF-MS to detect the hydrophilic small molecule metabolites of 13 kinds of cheese, and established a connection between this quantitative result and the sensory quality of cheese, providing a material target for the optimization of sensory quality in the future. Rodrigues et al.［9］ used NMR technology to comprehensively evaluate the fingerprint spectra of cheese containing prebiotics, and obtained a metabolite spectrum that could judge whether this type of cheese was mature by this method. Tian et al.［1］ from Shanghai Institute of Technology used gas chromatography-mass spectrometry (GC-MS), gas chromatography-olfactory (GC-O), sensory evaluation, aroma restructuring and aroma omission experiments to analyze the samples from the six most popular handmade milk fan workshops in Yunnan Province, China, and clarified the key flavor components［1］.

The study of Settachaimongkon et al.［10］ showed that after the co-inoculation of Streptococcus thermophilus, which has no proteolytic ability, and Lactobacillus delbrueckii subsp. Bulgaricus, into cow milk, both strains achieved higher growth, the milk acidification speed was faster, and the content of flavor metabolites of lactic acid bacteria was also higher. The study also showed that the metabolite spectra of microorganisms in the fermentation process could be used to judge the quality of solidified yogurt. Palomo et al.［11］ studied the characteristics of selenium-containing metabolites of lactic acid bacteria in selenium-enriched health yogurt, and showed that selenium was distributed in selenocysteine, thioredoxin, albumin, β-lactoglobulin and lactoperoxidase, and selenium might be related to the expression of heat shock protein and chaperone in Lactobacillus. With the wide application of metabolomics, the research on the determination of characteristic cheese biomarkers and specific metabolic profiles, as well as the relationship between these substances and lactic acid bacteria, will be more in-depth, so as to provide a theoretical basis for the scientific evaluation and in-depth utilization of lactic acid bacteria-fermented foods.

Application of Metabolomics in Fermented Vegetable Products

Fermented vegetables have a long shelf life, rich and unique taste, and have the effects of stimulating appetite, helping digestion and relieving greasiness. Lactic acid bacteria are widely used in the field of fermented vegetables in fermented foods. Lactobacillus is the more common lactic acid bacteria in fermented foods. The metabolism of lactic acid bacteria provides organic acids, amino acids, small peptides and some aromatic substances for fermented vegetables. Furthermore, lactic acid bacteria also have the function of inhibiting other bacteria and preventing vegetable spoilage in the process of vegetable fermentation. Combining nuclear magnetic resonance technology with high-throughput sequencing technology, Jung et al.［12］ studied the effects of Leuconostoc mesenteroides as a starter on the microbial flora and main flavor substances during the fermentation of Korean kimchi. The results showed that L. mesenteroides not only affected the change of the microbial phase during the fermentation of Korean kimchi, but also affected the production of some main flavor substances of fermented vegetables. Subsequently, another scholar also studied the role of red pepper powder in Korean kimchi by combining nuclear magnetic resonance technology with high-throughput sequencing technology, and the results showed that adding red pepper powder during the Kimchi fermentation process would prolong the fermentation lag period. Moreover, the abundance of Weissella was significantly higher than the other two lactic acid bacteria genera (Leuconostoc and Lactobacillus)［13］. Zhao et al.［14］ based on the global metabolomics strategy obtained the landmark magnetic flavor substances in the traditional old brine kimchi, and established the relationship between these target flavor substances and the lactic acid floras by target metabolomics. Liang et al.［5］ from Dalian University of Technology used Illumina HiSeq sequencing and GC-MS to reveal the effects of salt concentration on microbial diversity and volatile compounds during suancai fermentation. Studies have found that adding 6% (w/w) salt was very suitable for suancai fermentation and produced the best quality. Lactic acid bacteria and unclassified fungi were the main genus of suancai. 79 volatile compounds were detected during the fermentation of suancai. During the fermentation of suancai, volatile compounds were closely related to lactobacilli. Artificial inoculation of Lactobacillus plantarum could increase the volatile substances in the fermentation process of suancai. Xu et al.［15］ of China Agricultural University studied the correlation between the formation of fermented red pepper flavor and the growth and metabolism of microorganisms in the fermentation process. They applied high-throughput sequencing to analyze the changes in structures of bacterial and fungal communities during the fermentation process, and revealed that Staphylococcus was the main bacterial genus in the fermentation samples, while Hyphopichia and Kodamaea ohmeri were the most abundant fungi. Metabonomics analysis was used to study the correlation between non-cultured and purely cultured microorganisms and flavor compounds in fermentation. The study deepens our understanding of the microbiota involved in the production of fermented red peppers with unique flavor characteristics that were related to the core aroma.

Application of Metabolomics in Fermented Soy Products

Fermented soy products are traditional foods made from soybeans or black soybeans that are fermented by microorganisms. They have a long history in Asian countries such as China, Korea, and Japan. Fermented soy products are considered to be highly nutritious, and they have become more and more popular. The lactic acid bacteria involved in the fermentation process of soybean paste mainly include L. plantarum, Lactobacillus brevis, Lactobacillus sakei, Lactobacillus fermentum, and Tetracoccus halophilus. The metabolic activities of these lactic acid bacteria produce important flavor substances such as lactic acid, amino acids, monosaccharides and organic acids. Kim et al.［16］ conducted a global metabolomics study on the fermentation process of fermented soybean products through the combination of GC-TOF-MS and CE-TOF-MS, and the results revealed the global metabolic pathways of sugar and amino acid metabolism during the fermentation of soy products. Kang et al.［17］ monitored the metabolic accumulation process of the symbolic flavor substances during the fermentation of meju through UPLC-Q-TOF-MS, which provides a theoretical basis for the fermentation control of meju. Namgung et al.［18］ monitored the dynamic changes of metabolites during the fermentation of Korean traditional bean paste, and the results showed that during the fermentation process, the contents of 8 amino acids including alanine increased significantly after fermentation; the contents of palmitic acid and other 5 types of fatty acids were high in the whole fermentation process; and the organic acid metabolites mainly included carbonic acid, citric acid and lactic acid. Lee et al.［19］ used metabolomics to divide the fermentation of doenjiang into cooking, drying, fermentation, salt immersion and post-cooking, and investigated the change laws of monosaccharides, amino acids, fatty acids, isoflavones and soy saponins in different stages of fermentation. Wu et al.［2］ from Shenyang Agricultural University used meta-transcriptome sequencing, HS-SPME-GC-MS and amino acid analysis methods to analyze the changes of the soybean paste flora and metabolite profile over time, and identified 10 kinds of volatile compounds that contributed to the formation of soybean paste flavor. Moreover, the correlation between active microorganisms in soybean paste and physical and chemical properties and flavor substances was further analyzed. The results showed that Lactobacillus and Tetragenococcus were the core genera that affected color and flavor. This discovery provides new insights into the role of bacteria in the formation of fermented food flavor［2］.

Application of Metabonomics in Lactic Acid Bacteria-fermented Drinks

In addition to being widely used in fermented milk and fermented vegetables, lactic acid bacteria also play an important role in the fermentation of some fermented beverages and wines. Lee et al.［20］ used Oenococcus oeni in malolactic fermentation (MLF) of Meoru (Vitis coigneties), and analyzed the data with PCA and orthogonal partial least squares discriminant analysis (OPLS-DA). The results showed that there were no significant differences in the primary products from different O. oeni strains, but some important volatile flavor substances (2-phenylethyl alcohol, isoamyl alcohol, 2-butanol, ethyl caprylate, etc.) in its secondary metabolites had significant differences, and these flavor substances were closely related to wine quality［20］. In recent years, many researchers have used metabolome methods to study the effect of the interaction between yeast and lactic acid bacteria on the wine fermentation process. The results of Son et al.［21］ showed that after adding lactic acid bacteria to the Saccharomyces bayanus separate fermentation system, MLF caused decreases in the contents of malic acid and citric acid, and simultaneously increased the content of lactose; and the increase in the content of succinic acid indicated that S. bayanus inhibited MLF. Lopezrituerto et al.［22］ used 9 commercial wine samples as the objects of investigation to investigate the metabolome characteristics of ethanol fermentation and MLF metabolic process, and finally screened out isoamyl alcohol and isobutanol as the biomarkers for distinguishing the authenticity of La Rioja wine by combining MLF with the interval extended canonical variate analysis (iECVA) method. Li et al.［3］ from Northwest A&F University selected 4 kinds of lactic acid bacteria to carry out the lactic acid fermentation of Wada dates and muzao jujube, respectively, and analyzed the physicochemical and flavor quality of fermented jujube juice. It was concluded that the jujube juice could better improve the activity, quality and flavor of lactic acid bacteria after being fermented by lactic acid bacteria. Peng et al.［4］ from Northwest A&F University used 3 strains of compound lactic acid bacteria to study the fermentation of different varieties of apple juice, and studied the flavor and sensory characteristics of different apple juice after fermentation by lactic acid bacteria, and reasonable opinions were given on the suitability of processing apple varieties.

Guangpeng LIU et al. Application of Metabonomics in the Research of Lactic Acid Bacteria-fermented Foods

Conclusions

Metabolomics has been widely used in the field of lactic acid bacteria-fermented foods. Due to the complexity of lactic acid bacteria fermentation metabolites and the complexity of the fermented food systems involved, the current quality research and production technical specifications of lactic acid bacteria-fermented foods need to be further improved and perfected.  In order to deeply understand the ecological processes and functions of lactic acid bacteria in the food fermentation systems, and reveal the quality improvement mechanism of lactic acid bacteria-fermented foods, future studies of metabolomics in lactic acid bacteria-fermented foods should focus on the following aspects: ① Quenching causes the leakage of lactic acid bacterias intracellular metabolites, which will affect the accuracy of the data results. As a scientific researcher, we should optimize the each lactic acid bacteria quenching method, and establish a perfect and feasible quenching method. ② There is a lack of standardized and comprehensive metabolomics databases. Escherichia coli and Saccharomyces cerevisiae have established relatively complete metabolomics databases, and the metabolic databases of lactic acid bacteria need to be further improved. In order to systematically study biological sciences, a systematic and complete standardized database should be established, so as to improve the connection with other omics and to better interpret the relevant information of organisms, foods, etc. ③ In-depth study can be conducted on the metabolic mechanism of functional lactic acid bacteria from biomarkers to specific metabolic pathways. ④ Genomics, transcriptomics and proteomics can be combined to better interpret the impact of lactic acid bacteria on food quality and human probiotics.

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