2025 Volume 72 Issue 4 Article ID: 7204103
Perilla frutescens is a popular aromatic edible plant. The amount of Perilla residues produced by food or pharmaceutical industry is increasing because of the high demand for this plant. At present, most Perilla residues are incinerated, but there is increasing interest in using these materials as a biomass resource. In this study, an alkaline pretreatment to remove lignin from Perilla residues was optimized, and the ash, lignin, and total sugar contents of the treated materials were determined to evaluate their biomass potential. The optimum alkaline pretreatment for Perilla residues was 0.25 M NaOH at 121 °C for 60 min. The lignin and total sugar contents of the alkaline-pretreated Perilla residues were comparable to those reported for grain straw. These results suggest that alkaline-pretreated Perilla residues have high potential as biomass. With dual aims to reduce the volume of Perilla residues and to effectively use this resource, bacteria capable of decomposing Perilla seed shells after alkaline pretreatment were isolated from environmental samples. A total of 66 strains of degraders were isolated, of which one strain (strain SW8) was identified as Klebsiella aerogenes or Raoultella ornithinolytica with both cellulase and xylanase activities. Strain SW8 grew well at 25-35 °C with Perilla seed shells as the sole carbon source. Strain SW8 was identified as a useful bacterium to reduce the volume of, and effectively utilize, Perilla residues.
PSO, Perilla seed oil; CMC, carboxymethylcellulose; PCR, polymerase chain reaction
Perilla frutescens, in the Lamiaceae family, is widely distributed in East Asian countries such as Japan, China, Korea, and Vietnam. It has been used in Chinese medicine since ancient times and is cultivated as a food crop in mainland China, Japan, and Korea [1]. More recently, it has been used for both culinary and medicinal purposes not only in East Asia but also in North America and Europe [2]. Its leaves contain abundant polyphenolic compounds with high antioxidant potential. Perilla essential oil extracted from P. frutescens leaves exhibits high antioxidant, anticancer, anti-inflammatory, insecticidal, and antimicrobial activities [1]. Perilla seed oil (PSO) constitutes approximately 40 to 50 % of Perilla seeds [3], and the proportion of α-linolenic acid (54-64 %) in PSO is among the highest in the plant kingdom [4]. Polyunsaturated fatty acids, including α-linolenic acid, are essential for human health [3]. PSO is also rich in phytosterols, including β-amyrin, β-sitosterol, campesterol, and stigmasterol. The content of β-sitosterol is significantly higher in PSO than in other vegetable oils [5]. Furthermore, PSO is rich in tocopherols, and has been reported to exhibit multiple bioactivities including antioxidant, anti-inflammatory, hypolipidemic, hypoglycemic, neuroprotective, and immunomodulatory activities [6, 7]. The global market for PSO is predicted to increase to $2,415.6 million by 2031, highlighting the strong market potential for Perilla and its derived products [2]. In addition to its functional oils, other extracts of P. frutescens have been shown to have bioactive properties, such as anti-allergy [8], anti-inflammatory [9], antioxidant [10], antibacterial [11], and antidepressant properties [12].
The amount of Perilla residues (leaf and stem ends, seed shells after oil extraction) produced by food or pharmaceutical industry is increasing as a result of the increasing demand for P. frutescens, although unfortunately there is no statistical data that accurately quantifies these materials. At present, most Perilla residues are incinerated. Therefore, reducing the amount of Perilla residues and finding effective ways to use these materials is attracting worldwide attention. Additionally, the production of high-value-added products from lignocellulosic biomass has attracted great interest in recent years [13, 14]. Special emphasis has been placed on polysaccharides, which are abundant in plant materials. For example, the use of polysaccharides in fermentation systems to produce bioethanol, bulk and fine chemicals, polyester-based materials, additives, hydrogels, and pharmacological products has been reported [13, 14]. However, the processing of plant biomass into fermentable sugars by bacteria and their associated enzymes is hampered by the complexity of the secondary cell wall structure and the presence of lignin [15]. Alkaline pretreatments have been shown to promote the degradation of lignocellulosic agricultural wastes such as grain straw [16]. Alkaline environments remove uronic acids and acetyl groups from polysaccharides, making them more accessible to enzymes for further hydrolysis, reducing cellulose crystallinity and increasing its biodegradability [17, 18]. However, to our knowledge, the biomass potential of Perilla residues has not been determined, and bacteria capable of degrading these materials have not been isolated. In this study, the amounts of ash, lignin, and total sugars in Perilla residues were determined. Subsequently, the conditions of an alkaline pretreatment for lignin removal were optimized. Bacteria capable of decomposing Perilla seed shells after alkaline pretreatment were isolated from environmental samples, and the possibility of utilizing one of the bacterial strains to reduce the volume and effectively use Perilla seed shell residue was examined.
Perilla frutescens. Perilla residues (leaf and stem ends, seed shells after oil extraction) were obtained from a plant factory at Ushidake, Toyama, Japan. These materials were dried naturally and pulverized using a blender (Model 7011HB, Waring Commercial, Boston, MA, USA) prior to pretreatment.
Total sugar, ash, and lignin contents of Perilla residues. The composition of Perilla residues was determined using the Klason lignin method [19], with modifications. First, 0.3 g Perilla residue was soaked in 4.5 mL 72 % sulfuric acid for 3 h, and diluted to 3 % w/v with water. The mixture was then autoclaved at 121 °C for 30 min, and after cooling, the solid material was removed using a glass fiber filter (Advantec Ltd., Tokyo, Japan). The liquid was used for sugar analysis. The total sugar content in the liquid phase was determined using the phenol-sulfuric acid method. The calibration curve was constructed using D-glucose. Another 0.3 g Perilla residue was incinerated at 500 °C for 2 h and the ash weight was determined. The lignin content (the others) was calculated by subtracting the weight of ash and total sugars from the initial dry weight. All the data are shown as the means ± SDs (N = 3).
Optimization of alkaline pretreatment of Perilla residues. An alkaline pretreatment can remove lignin from plant materials [20, 21], so we optimized the conditions of this pretreatment for Perilla residues. The residues were immersed in various concentrations of NaOH solution and then heated to various temperatures for a range of times. The treated material was neutralized with 1 M HCl solution after cooling and then washed thoroughly several times with distilled water. The washed materials were completely dried in a SANYO Convection oven MOV-212F(U) (SANYO Electric Co., Ltd., Osaka, Japan) at 50 °C before composition analysis, as described above. To determine the optimum NaOH concentration for the alkaline pretreatment, the Perilla residues were treated with 0.125 M, 0.25 M, or 0.5 M NaOH solution overnight and then heated at 121 °C for 60 min. To determine the optimum treatment temperature, the Perilla residues were treated with 0.25 M NaOH solution overnight and then heated to 80, 121, or 135 °C for 60 min. To determine the optimum treatment time, the Perilla residues were treated with 0.25 M NaOH solution overnight and then heated to 121 °C for 10, 30, or 60 min.
Isolation of bacteria capable of degrading Perilla residues. Various environmental samples (soil, seawater, river water, snow) were collected from several sites in Toyama Prefecture, Japan. After transport to the laboratory in an icebox, the samples were prepared for analysis. The primary medium used in this study was M9 medium (per L distilled water: 6.8 g Na2HPO4, 3.0 g KH2PO4, 0.5 g NaCl, 1.0 g NH4Cl, 2 mL 1 M MgSO4, 0.1 mL 1 M CaCl2), which has no carbon source to support bacterial growth. To isolate bacteria capable of degrading Perilla residues, 0.1 g (wet weight) of each sample was transferred to a conical beaker containing 100 mL M9 medium and alkaline-pretreated dried Perilla seed shells (0.5 % w/v). The mixture was then incubated on a rotating shaker (Bioshaker BR-180LF; Titec Corporation, Saitama, Japan) at 25 °C with shaking at 140 rpm for 7 days. When fragmentation of Perilla seed shell was visually observed, a portion of the culture was spread on solid M9 plates (agar, 1.5 % w/v). Individual colonies were then picked from the plates and inoculated into M9 liquid medium containing Perilla seed shell fragments (0.5 % w/v) and incubated with shaking for 2 to 3 days. Bacterial clones observed to degrade Perilla seed shell fragments were stored on M9 agar slants or plates for further use.
The cellulase and xylanase activities of the isolated bacteria were evaluated by halo assays using M9 agar medium containing 0.5 % (w/v) carboxymethylcellulose (CMC; Sigma-Aldrich, St. Louis, MO, USA) or xylan from corn core (Tokyo Chemical Industry Co., Ltd., Tokyo, Japan) [22]. Bacterial strains were cultured for 7 days on agar plates at 25 °C, then the plates were stained with 0.1 % w/v Congo red solution for 20 min. Finally, the stained plates were decolorized with 1 M NaCl solution for 20 min to confirm the halo.
The isolated bacteria were identified by sequencing 16S rDNA. Genomic DNA was extracted from each bacterial colony, and 16S rDNA was amplified by polymerase chain reaction (PCR) using the eubacterial universal primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-TACGGYTACCTTGTTACGACTT-3′). Each PCR sample contained 1×Ex Taq buffer, 200 μM dNTP mix, 0.25 μM of each primer, and 0.5 U Ex Taq HS (Taq DNA polymerase; Takara Bio Inc., Shiga, Japan). The amplified products were purified with the QIAquick PCR Purification Kit (Qiagen K.K., Tokyo, Japan), and then directly sequenced using the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA), an ABI Prism 3130xl Genetic Analyzer (Applied Biosystems), and the primers r1L, r2L, r3L, r4L, f1L, f3L, and 926f [23]. The BLAST program on the NCBI website was used to search for sequences homologous to those of the isolates (http://www.ncbi.nlm.nih.gov).
Bacteria capable of degrading Perilla residues were cultured in 100 mL M9 medium containing dried alkaline-pretreated dried Perilla seed shells (0.5 % w/v). Bacterial cells were inoculated at concentrations of approximately 106 cells/mL followed by incubation at 15 to 45 °C with shaking at 140 rpm for 24 h. Bacterial growth was estimated by counting under a microscope using a bacterial counting chamber (Erma Inc., Saitama, Japan).
Nucleotide sequence accession number. The GenBank/EMBL/DDBJ accession number of the nucleotide sequence of the 16S rRNA gene of strain SW8 reported in this paper is LC871423.
Lignocellulosic biomass has been in the research spotlight because of its potential use in the production of sustainable and renewable energy and fuels. In Europe, agricultural and residual lignocellulosic biomass is considered an important resource for the expansion of biogas and biomethane production [24]. The volume of those materials is expected to double by 2030 compared with 2015 [24]. Various straws (wheat straw, barley straw, rye straw, vegetable straw) are important sources of agricultural and residual lignocellulosic biomass [25, 26]. However, the biomass potential of P. frutescens, which has recently attracted attention as food and medicine, has not been investigated.
As Perilla production increases in the future, the amount of waste will also increase, so it is important to analyze its components. First, the contents of ash, total sugars (sugar extracted), and lignin (others) in dried Perilla residues (leaf ends, stem ends, seed shells) were examined (Table 1). The contents of ash, total sugars (sugar extracted), and lignin (others) in leaf ends were 0.180 ± 0.003 g/g-Perilla residues (18.1 ± 0.4 %), 0.227 ± 0.01 g/g-Perilla residues (22.5 ± 1.1 %), and 0.593 ± 0.007 g/g-Perilla residues (59.4 ± 0.5 %), respectively; those in stem ends were 0.233 ± 0.003 g/g-Perilla residues (23.4 ± 0.5 %), 0.323 ± 0.013 g/g-Perilla residues (32.3 ± 1.4 %), and 0.443 ± 0.017 g/g-Perilla residues (44.2 ± 1.7 %), respectively; and those in seed shells were 0.063 ± 0.003 g/g-Perilla residues (6.35 ± 0.2 %), 0.230 ± 0.020 g/g-Perilla residues (23.0 ± 1.8 %), and 0.707 ± 0.017 g/g-Perilla residues (70.7 ± 1.6 %), respectively. The lignin contents of wheat straw, barley straw, rye straw, and rape straw grown in experimental fields at the University of Copenhagen were reported to be 17.6 ± 0.5 %, 16.8 ± 0.3 %, 16.5 ± 0.3 %, and 15.4 ± 0.4 %, respectively [25]; and those of Thai and Egyptian rice straw were reported to be 15.33 ± 1.18 % and 36.1 %, respectively [27, 28]. The total sugar contents in those materials were also very high (50-80 %) [25, 27, 28]. Thus, compared with other types of biomass, the lignin content of the Perilla residue was high, and it seemed to inhibit the sugar extraction. Therefore, it was considered that pretreatment to remove lignin which was expected to hinder the extraction of sugar was necessary.
Table 1. Composition of P. frutescens residues.
| leaf ends | stem ends | seed shells | ||
| Ashes | (g/g-Perilla residues) | 0.180 ± 0.003 | 0.233 ± 0.003 | 0.063 ± 0.003 |
| (%) | 18.1 ± 0.4 | 23.5 ± 0.5 | 6.35 ± 0.2 | |
| Sugar extracted | (g/g-Perilla residues) | 0.227 ± 0.01 | 0.323 ± 0.013 | 0.230 ± 0.02 |
| (%) | 22.5 ± 1.1 | 32.3 ± 1.4 | 23.0 ± 1.8 | |
| Others※ | (g/g-Perilla residues) | 0.593 ± 0.007 | 0.443 ± 0.017 | 0.707 ± 0.017 |
| (%) | 59.4 ± 0.5 | 44.2 ± 1.7 | 70.7 ± 1.6 | |
※Others contain lignin and unextracted sugars.
Alkaline pretreatment has been shown to facilitate the degradation of lignocellulosic agricultural wastes such as grain straw [16]. In this study, alkaline pretreatment conditions for lignin removal from Perilla seed shells were optimized (Fig. 1). When Perilla seed shells were treated with 0.125 M, 0.25 M, and 0.5 M NaOH, the lignin contents (the others) of the treated materials were 0.537 ± 0.067 g/g-Perilla residues (53.6 ± 6.5 %), 0.430 ± 0.023 g/g-Perilla residues (42.9 ± 2.3 %), and 0.420 ± 0.057 g/g-Perilla residues (41.9 ± 5.6 %), respectively; and the total sugar contents (sugar extracted) were 0.357 ± 0.037 g/g-Perilla residues (35.8 ± 3.8 %), 0.460 ± 0.030 g/g-Perilla residues (46.0 ± 2.6 %), and 0.470 ± 0.013 g/g-Perilla residues (47.1 ± 0.5 %), respectively (Table 2). When the alkaline pretreatment of Perilla seed shells was conducted at 80, 121, and 135 °C, the lignin contents (the others) of the treated materials were 0.533 ± 0.047 g/g-Perilla residues (53.2 ± 4.6 %), 0.430 ± 0.023 g/g-Perilla residues (42.9 ± 2.3 %), and 0.447 ± 0.027 g/g-Perilla residues (44.7 ± 2.6 %), respectively, and the total sugar contents (sugar extracted) were 0.360 ± 0.043 g/g-Perilla residues (36.0 ± 4.9 %), 0.460 ± 0.033 g/g-Perilla residues (46 ± 2.6 %), and 0.433 ± 0.017 g/g-Perilla residues (43.2 ± 1.4 %), respectively (Table 2). When the heating time during the pretreatment of Perilla seed shells was 10, 30, and 60 min, the lignin contents (the others) of the treated materials were 0.477 ± 0.030 g/g-Perilla residues (47.7 ± 2.9 %), 0.473 ± 0.013 g/g-Perilla residues (47.4 ± 1.4 %), and 0.410 ± 0.023 g/g-Perilla residues (43.1 ± 0.2 %), and the total sugar contents (sugar extracted) were 0.430 ± 0.033 g/g-Perilla residues (43.1 ± 2.8 %), 0.440 ± 0.017 g/g-Perilla residues (44.1 ± 1.2 %), and 0.457 ± 0.017 g/g-Perilla residues (45.8 ± 0.6 %), respectively (Table 2). On the basis of these results, the optimum NaOH concentration, reaction temperature, and treatment time for alkaline pretreatment of Perilla seed shells were determined to be 0.25 M, 121 °C, and 60 min, respectively.
Strain SW8 was cultured on M9 agar medium containing 0.5 % (w/v) carboxymethylcellulose or xylan at 25 °C for 7 days and then halos were detected. (A) Cellulase activity. (B) Xylanase activity.
Table 2. Composition of Perilla seed shells after alkaline pretreatment under various conditions.
| NaOH concentration | Reaction temperature | Treatment time | ||||||||
| 0.125 M | 0.25 M | 0.5 M | 80 °C | 121 °C | 135 °C | 10 min | 30 min | 60 min | ||
| Ashes | (g/g-Perilla residues) | 0.107 ± 0.030 | 0.110 ± 0.003 | 0.110 ± 0.060 | 0.107 ± 0.003 | 0.110 ± 0.003 | 0.120 ± 0.013 | 0.093 ± 0.003 | 0.087 ± 0.003 | 0.110 ± 0.003 |
| (%) | 10.6 ± 2.9 | 11.1 ± 0.4 | 10.9 ± 6.0 | 10.7 ± 0.5 | 11.1 ± 0.4 | 12.1 ± 1.2 | 9.18 ± 0.3 | 8.53 ± 0.5 | 11.1 ± 0.4 | |
| Sugar extracted | (g/g-Perilla residues) | 0.357 ± 0.037 | 0.460 ± 0.030 | 0.470 ± 0.013 | 0.360 ± 0.043 | 0.460 ± 0.033 | 0.433 ± 0.017 | 0.430 ± 0.033 | 0.440 ± 0.017 | 0.457 ± 0.017 |
| (%) | 35.8 ± 3.8 | 46.0 ± 2.6 | 47.1 ± 0.5 | 36.0 ± 4.9 | 46.0 ± 2.6 | 43.2 ± 1.4 | 43.1 ± 2.8 | 44.1 ± 1.2 | 45.8 ± 0.6 | |
| Others※ | (g/g-Perilla residues) | 0.537 ± 0.067 | 0.430 ± 0.023 | 0.420 ± 0.057 | 0.533 ± 0.047 | 0.430 ± 0.023 | 0.447 ± 0.027 | 0.477 ± 0.030 | 0.473 ± 0.013 | 0.410 ± 0.023 |
| (%) | 53.6 ± 6.5 | 42.9 ± 2.3 | 41.9 ± 5.6 | 53.2 ± 4.6 | 42.9 ± 2.3 | 44.7 ± 2.6 | 47.7 ± 2.9 | 47.4 ± 1.4 | 43.1 ± 0.2 | |
※Others contain lignin and unextracted sugars.
The composition of leaf ends, stem ends, seed shells subjected to the optimal alkaline pretreatment is shown in Table 3. The lignin content (the others) in the Perilla leaf ends after alkaline pretreatment was comparable to those previously reported for wheat straw, barley straw, rye straw, and rape straw [25]. The total sugar contents (sugar extracted) of leaf ends, stem ends, and seed shells after alkaline pretreatment were also comparable to those previously reported for other biomass materials [25, 27, 28]. These results suggest that alkaline-pretreated Perilla residues have high potential as a biomass resource.
Table 3. Composition of P. frutescens after alkaline pretreatment.
| leaf ends | stem ends | seed shells | ||
| Ashes | (g/g-Perilla residues) | 0.167 ± 0.010 | 0.133 ± 0.030 | 0.110 ± 0.003 |
| (%) | 16.8 ± 1.1 | 13.3 ± 2.9 | 11.1 ± 0.4 | |
| Sugar extracted | (g/g-Perilla residues) | 0.693 ± 0.103 | 0.417 ± 0.023 | 0.460 ± 0.030 |
| (%) | 69.5 ± 10.2 | 41.7 ± 2.4 | 46.0 ± 3.2 | |
| Others※ | (g/g-Perilla residues) | 0.137 ± 0.003 | 0.450 ± 0.040 | 0.430 ± 0.023 |
| (%) | 13.7 ± 4.5 | 44.9 ± 4.2 | 42.9 ± 2.3 | |
※Others contain lignin and unextracted sugars.
With dual aims to reduce the volume of Perilla residues and effectively use this resource, we isolated bacteria capable of decomposing Perilla seed shells after alkaline pretreatment from environmental samples. A total of 66 bacterial strains capable of degrading pretreated Perilla seed shells were isolated from various environmental samples. Each isolate was cultivated in a medium containing Perilla seed shells as the sole carbon source. Of the isolates examined, strain SW8 isolated from seawater was the only one in which both cellulase and xylanase activities were detected by halo assays (Fig. 1). The 1,462-bp 16S rDNA sequence of strain SW8 was used to identify the species. The sequence showed 99.9 % similarity with Klebsiella aerogenes strain MAE1 (GenBank accession number PQ432925) and with Raoultella ornithinolytica strain FMC41 (GenBank accession number KF358448). Klebsiella aerogenes strain MAE1 was isolated at Kalasin University, Thailand, but detailed information has not been registered. Raoultella ornithinolytica strain FMC41 was isolated from the gut contents of grass carp [29]. Raoultella ornithinolytica, previously thought to be a relative of K. oxytoca, was classified as R. ornithinolytica in 2001 because of biochemical and genetic differences [30]. Sequence analysis of 16S rDNA alone did not reveal which bacteria were more closely related because they showed identical homology. Further analysis such as analysis of biochemical properties of strain SW8 and DNA-DNA hybridization between strain SW8 and both strains is necessary in order to identify the species accurately in the future. To our knowledge, the isolation of R. ornithinolytica or K. aerogenes with xylanase activity has not been reported previously. Therefore, strain SW8 is considered to be the first K. aerogenes or R. ornithinolytica strain with xylanase activity. In further research, it will be useful to isolate the gene(s) encoding xylanase(s) in strain SW8 using the shotgun cloning method, and carry out detailed characterization of this strain.
The growth of strain SW8 was examined in the range of 15-45 °C (Fig. 2A). It did not grow at 15 and 45 °C, but grew well at 25 °C and showed the fastest growth at 35 °C (Fig. 2A). Strain SW8 did not grow on M9 medium without Perilla seed shells (Fig. 2B). The fact that strain SW8 was able to utilize Perilla seed shells as a carbon source suggests that it will be useful for reducing the volume of this waste stream. At present, there is a focus to use bacteria that grow at ambient temperatures to utilize straw residues as biomass, as this simplifies production systems and reduces costs [31, 32]. Strain SW8 grew well in the range of 25 to 30 °C, so it will be a useful bacterium to reduce the volume and effectively use Perilla residues.
(A) Strain SW8 was cultured in M9 medium containing 0.5 % (w/v) alkaline-pretreated Perilla seed shells at 15-45 °C for 24 h. (B) Strain SW8 was cultured in M9 medium with or without 0.5 % (w/v) alkaline-pretreated Perilla seed shells at 35 °C for 24 h.
In this study, the optimum conditions for alkaline pretreatment of Perilla residues were determined as follows: NaOH concentration: 0.25 M, reaction temperature: 121 °C, reaction time: 60 min. The total sugar (sugar extracted) and lignin (the others) contents of the alkaline-pretreated Perilla residues were comparable to those previously reported for other types of straw biomass. These results suggest that alkaline-pretreated Perilla residues have high potential as a biomass resource. With aims to reduce the volume of Perilla residues and effectively utilize this resource, we isolated bacteria capable of decomposing pretreated Perilla seed shells from environmental samples. A total of 66 strains capable of degrading pretreated Perilla seed shells were isolated, one of which (strain SW8) was identified as K. aerogenes or R. ornithinolytica with both cellulase and xylanase activities. Strain SW8 grew well at 25 to 35 °C with Perilla seed shells as the sole carbon source. Therefore, strain SW8 was screened as a useful bacterium for volume reduction and effective utilization of Perilla residues.
The authors declare no competing financial interests.
This study was supported in part by grants to A.S. (KAKENHI grant nos. 20K06203 and 23H02291 from JSPS). We also thank Edanz for editing a draft of this manuscript. Generative AI was not used to write this paper.
