Dietary supplementation of glycerol monolaurate improves growth performance, meat quality, and gut microbiota in black pigs

Abstract

This study investigated the effects of glycerol monolaurate (GML) on growth performance, meat quality, and gut microbiota in black pigs. A total of 135 castrated Dongliao black pigs (age, 170 ± 5 d; initial body weight, 82.41 ± 1.65 kg; male:female = 72:63) were weighed and randomly assigned into three groups (3 replicates/group, 15 pigs/replicate). Pigs were fed either a basal diet (control group) or the basal diet supplemented with 250 and 750 mg/kg GML for 56 d. The results showed that GML supplementation linearly increased final body weight (P = 0.031) and quadratically changed the feed conversion ratio (P = 0.025) without causing glucolipid metabolism disorders. In addition, GML improved meat quality and promoted protein (P = 0.034) and amino acid (AA) deposition (P = 0.005) in the muscle. These beneficial effects were attributed to improvements in the intestinal micro-environment, as GML significantly increased the relative abundances of Terrisporobacter and Romboutsia (P < 0.05), and elevated the concentration of total bile acids related to gut microbiota (P = 0.001). Overall, these findings demonstrate the potential of GML as a functional feed additive to improve growth performance and meat quality of finishing pigs, with 750 mg/kg GML showing greater efficacy than 250 mg/kg.

1. Introduction

The Dongliao black pig is an indigenous Chinese breed that is highly favored by consumers due to its excellent meat quality (Gao et al., 2024). However, compared with the Duroc × Landrace × Yorkshire (DLY) crossbred pigs, Dongliao black pigs have a slower growth rate, higher feed intake, and reduced feed efficiency, making it difficult to gain a competitive production efficiency advantage in China’s swine industry. The market age of DLY pigs is about 6 months, whereas Dongliao black pigs have a market age ranging from 8 to 10 months which increases breeding herd to market weight throughput and production costs (Guo et al., 2022). Nutritional interventions to improve growth rate and feed efficiency of Dongliao black pigs without compromising meat quality are therefore needed. An improper balance of nutrients and choice of feed ingredients and feed additives can result in less desirable meat quality (Xu et al., 2022).

Glycerol monolaurate (GML) is a medium-chain fatty acid glyceride formed from glycerol and lauric acid (C12:0). It is naturally present in breast milk, coconut oil, and palm oil (Kong et al., 2023). As a common food additive, GML is mainly used to improve food quality, extend shelf life, and promote food stability in the food industry. In addition to the well-known antibacterial and emulsifying properties, GML also regulates intestinal health, immune defense, glucolipid metabolism, and bone growth (Chen et al., 2022; Luo et al., 2022). Recently, in the Penaeus vannamei aquaculture, GML supplementation significantly improved weight gain rate and reduced a feed conversion ratio (FCR) (Liu et al., 2024). In our previous studies, GML supplementation significantly increased body weight, crude protein (CP) content, and unsaturated fatty acids (UFA) content of large yellow croaker (Zhuang et al., 2022). Moreover, we also found that both chicken broth and meat taste were improved by GML supplementation, which might be related to the increase in amino acid (AA) content in the muscle of chicken breast (Liu et al., 2021a, Liu et al., 2021b). In pig husbandry, maternal supplementation with GML significantly increased villi height and crypt depth in the ileum and jejunum of piglets, and reduced the relative abundance of Escherichia shigella, suggesting enhanced nutrient digestion and absorption, and gut microbiota regulation (Zhao et al., 2023). Collectively, these findings demonstrate the potential of GML to promote animal growth, regulate intestinal health and improve meat quality (Kong et al., 2022). Unlike sows and piglets, the primary objectives in finishing pig production are to maximize growth rate and optimize meat quality. However, the relatively slow growth rate and poor feed efficiency of black pigs remains major challenges. Based on the potential biological activities of GML mentioned above, we hypothesized that dietary GML supplementation could enhance growth rate and FCR in finishing black pigs by modulating the gut microbiota, without affecting meat quality.

This study aimed to investigate the effects of dietary GML on growth performance, serum biochemistry, and meat quality in finishing Dongliao black pigs. Furthermore, it was analyzed the potential underlying mechanisms from the perspective of gut microbiota, aiming to provide a comprehensive understanding and potential application of GML in the pig industry and other aquaculture industries.

2. Materials and methods

2.1. Animal ethics statement

The experimental procedures were approved by the Institutional Animal Ethics Committee of Zhejiang University (approval number ZJU20240866).

2.2. Experimental reagents and animal experiment

Glycerol monolaurate with 95% purity was obtained from Hangzhou Longyu Biotechnology Co., Ltd. (Hangzhou, Zhejiang, China). Porcine glucagon like peptide 1 (GLP-1), porcine peptide YY (PYY), porcine neuropeptide Y (NPY), and porcine ghrelin enzyme-linked immunosorbent assay (ELISA) kits (JYM0219Po, JYM0390Po, JYM0389Po, and JYM0232Po, respectively) were purchased from Wuhan Jiyinmei Biotechnology Co., Ltd. (Wuhan, Hubei, China). Glucose, total triglyceride (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (AKP), blood urea nitrogen (BUN), creatinine (CRE) and total bile acid (TBA) assay kits (F006-1-1, A110-1-1, A111-1-1, A113-1-1, A112-1-1, C009-2-1, C010-2-1, A059-2-2, C013-2-1, C011-2-1, and E003-2-1, respectively) were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, Jiangsu, China).

The Bicinchoninic acid assay (BCA) protein quantitative kit (P0010) was purchased from Beyotime Biotechnology Co., Ltd. (Shanghai, China). Other chemical reagents were obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China), such as petroleum ether, sulfuric acid, copper sulfate, potassium sulfate, anhydrous ethanol, acetyl chloride, methanol, potassium hydroxide, n-hexane, sodium sulfate, sodium chloride, and sodium hydroxide.

The experiment was performed at the Shaoxing Tiansheng Farming Co., Ltd. (Shaoxing, Zhejiang, China). A total of 135 castrated Dongliao black pigs (age, 170 ± 5 d; initial body weight, 82.41 ± 1.65 kg; male:female = 72:63) were weighed and randomly assigned into three groups based on a complete randomized block design using sex and initial body weight as blocks (3 pens/group, 15 pigs/pen, males:female = 8:7). Pigs were housed in an environmentally controlled facility (temperature 24 ± 1 °C; humidity [65 ± 5]%) with concrete-floored pens (5.0 m × 4.0 m). Each pen contained 15 pigs (stocking density 1.3 m²/head), equipped with a stainless-steel feeder and three cup drinkers. The photoperiod was maintained at 14 h light-10 h dark. All pigs were allowed to eat and drink freely, and each pen had an independent feed delivery system to accurately calculate feed consumption. To ensure uniform distribution of GML within the basal diet, GML was first thoroughly mixed with the premix feed (4% in the basic diet) before being blended with the basal diet, and put in bags labeled as treatment 1, 2, and 3. Based on previous studies and application potential, an effective dose of GML in pig trials is around 200 to 1000 mg/kg (Zhao et al., 2023), so 250 and 750 mg/kg were chosen. The group information is as follows: control group (NCD), basal diet; GML250 group (GML250), basal diet with 250 mg/kg of GML group; GML750 group (GML750), basal diet with 750 mg/kg of GML.

Following a 3-d adaptive phase, the experimental period lasted for 53 d, making the entire experimental duration 56 d. Throughout this period, the health conditions of the pigs were closely monitored. No clinical signs of disease or mortality were observed in any pigs throughout the experimental period.

Growth performance included initial weight, final weight, net weight gain, average daily weight gain (ADG), average daily feed intake (ADFI), FCR, carcass yield, and backfat thickness. Growth performance and feed intake were calculated based on each pen (n = 3 pen/group). Carcass yield and backfat thickness were calculated based on individual pigs (n = 6 pig/group).

The specific formulas were as follows:

ADG (g/d) = Total weight gain (g)/Experimental days (d);

ADFI (kg/d per pig) = Total feed intake/Experimental days/The number of pigs per replicate;

FCR = ADFI/ADG.

Backfat thickness was the height of the fat at the back between the sixth and seventh ribs in the front row. Carcass yield was the ratio of hot carcass weight to fresh weight (excluding head, hooves, tail, hair, blood, and internal organs). At the end of the feeding trial, the pigs were fasted overnight and then were slaughtered by electrical stunning, exsanguinated, dehaired, peeled, eviscerated, and split down the midline according to the Chinese guidelines (China National Standard, 2019) on the next day after weight measurement. To reduce stress response, pigs (average body weight, 106−110 kg) were selected for blood collection. Blood samples (10 mL) were collected from the anterior vena cava of each pig at 05:00 using sterile syringes after fasting but before slaughtering. The supernatant of the blood was obtained by centrifugation at 3000 × g for 15 min at 4 °C. Sections of liver (50 g) and fresh rectal feces (20 g) from each pig (n = 8 pigs/group) were collected, and all samples were frozen with liquid nitrogen. Meanwhile, the carcass weight and backfat thickness were measured after slaughter for an instant measurement. Fresh longissimus thoracis (LT; 100 g) between the 13th and 14th thoracic vertebrae of each pig (n = 6 pigs/group) was collected, and all samples were stored at −80 °C.

2.3. Determination of feed composition

Basal diets were formulated according to nutrient requirements for finishing black pigs. The composition and nutrient levels of basal diets are shown in Table 1. The CP was analyzed by the Kjeldah apparatus (B-324, Büchi Labortechnik AG, Flawil, Switzerland) according to the GB/T 6432-2018 (China National Standard, 2018a). The crude fiber, crude ash, and total calcium were analyzed by the high-temperature calcination method via a muffle furnace (SX2-15-10, Honglang Instrument Co., Ltd., Zhengzhou, Henan, China) according to the GB/T 6434-2022 (China National Standard, 2022), GB/T 6438-2007 (China National Standard, 2007), and GB/T 6436-2018 (China National Standard, 2018b), respectively. Total phosphorus was analyzed using a spectrophotometer (UV2006 PC, Shimadzu Ltd., Kyoto, Japan) according to the GB/T 6437-2018 (China National Standard, 2018c). The feed was ashed by high-temperature calcination via a muffle furnace, and the residue was dissolved in hydrochloric acid. Then, total copper was analyzed by an atomic absorption spectrophotometer (AA-7000, Shimadzu Ltd., Kyoto, Japan) according to the GB/T 13885-2017 (China National Standard, 2017). Feeds were hydrolyzed into AA under the action of 6 mol/L hydrochloric acid, and Lys was measured using an automatic AA analyzer (L-8900, Hitachi High Tech, Tokyo, Japan) after separation by ion exchange chromatography and derivatization via an indenone column. Organic matter is calculated through the formula:

Table 1.

The composition and nutrient levels of basal diets (% DM basis).

Ingredients	Content	Nutrient levels2	Content
Corn	9.5	Digestible energy, MJ/kg	12.03
Wheat	10.0	CP	13.00
Brown rice	10.0	Crude fiber	10.00
Barley	15.0	Crude ash	8.00
Flour	8.5	Calcium	0.70
Barley roots	12.0	Phosphorus	0.25
Alfalfa	9.0	Sodium chloride	0.60
Yellow wine lees	8.0	Copper, mg/kg	25.00
Rice bran meal	8.0	Lys	0.95
Corn germ meal	6.0	ADF	8.28
Premix1	4.0	NDF	19.03
Total	100.0	Organic matter	90.40

DM = dry matter; CP = crude protein; ADF = acid detergent fiber; NDF = neutral detergent fiber.

¹Premix provided the following per kg of the diet: Cu (CuCl₂) 10 mg, Fe (FeSO₄·7H₂O) 70 mg, Zn (ZnSO₄·7H₂O) 20 mg, Mn (MnSO₄·H₂O) 10 mg, I [Ca(IO₃)₂] 1 mg, Se (Na₂SeO₃) 0.3 mg, Co (CoSO₄·7H₂O) 0.6 mg, vitamin A (retinyl acetate) 3000 IU, vitamin D₃ (cholecalciferol) 1050 IU, vitamin E (DL-α-tocopheryl acetate) 200 mg, vitamin K (menadione sodium bisulfite complex) 10.4 mg, vitamin B₁ (thiamine mononitrate) 8.5 mg, vitamin B₂ (riboflavin) 21.1 mg, vitamin B₆ (pyridoxine hydrochloride) 11.7 mg, vitamin B₁₂ (cyanocobalamin) 0.1 mg, biotin (vd-biotin) 0.52 mg, folic acid 3.2 mg, niacin (nicotinamide) 101 mg, pantothenic acid (D-calcium pantothenate) 55.2 mg, choline (choline chloride) 300 mg.

²Digestible energy, ADF and NDF were calculated from data provided by China Feed Database (2020).

Organic matter = 100 − Ash content − Moisture content.

2.4. Biochemical assays

The concentrations of GLP-1, PYY, NPY, and Ghrelin were measured using ELISA kits according to the manufacturer’s protocols. The concentrations of glucose, TC, TG, LDL-C, HDL-C, ALT, AST, AKP, BUN, CRE, and TBA were analyzed using commercial reagent kits, and was measured by a using microplate reader (Infinite M200 Pro Tecan Laboratory Equipment Co., Ltd., Shanghai, China).

2.5. Lipid analysis

The liver tissue or feces (0.1 g) was measured, and 1 mL of a chloroform-methanol mixture (2:1, v/v) was added to facilitate tissue homogenization. The homogenized mixture was then incubated on a temperature-controlled shaker for 6 h. After incubation, the sample was centrifuged at 13,000 × g for 15 min at 4 °C to separate the supernatant. The total lipids were subsequently extracted by evaporating the solvent and weighed.

Hepatic lipid (0.04 g) was dissolved in 2 mL of n-hexane, and 0.2 mL of potassium hydroxide-methyl alcohol solution (2 mol/L) was added. After homogeneous mixing, 1 g of sodium sulfate was added to obtain the upper organic phase. Then, the organic phase was collected and filtered for gas chromatography analysis. Specific instrument parameters were the same as the previous work (Liu et al., 2020a).

2.6. Meat quality

Crude protein, crude fat, moisture, ash, and fatty acid composition of LT were analyzed according to the previous study (Liu et al., 2020a). Amino acid composition was measured via an automatic AA analyzer (L-8900).

Meat color parameters, encompassing lightness (L∗), redness (a∗), and yellowness (b∗), were quantified using a Minolta Chromameter (CR400-410, Minolta Sensing Inc., Osaka, Japan). The device had a 2° observation angle and a 50-mm measurement aperture, and was operated under D65 standard light source conditions. To ensure measurement accuracy, a white reference plate served as the device’s calibration reference before each analysis. Color readings were recorded after a 1-h blooming period to optimize measurement reliability and precision.

A calibrated portable pH meter (Orion 4 starA211, Thermo Fisher Scientific Inc., Waltham, MA, USA) was used to measure the post-mortem pH values at 24 h. Instrument calibration was performed using standardized buffer solutions at pH 4.0 and 7.0, and the entire process was conducted at a controlled temperature of 25 °C to eliminate temperature-related variability.

To assess drip loss, LT tissue was prepared into three cubes (6 cm × 4 cm × 4 cm) and suspended at 4 °C for 24 h, with quantitative analysis performed using the weight loss method.

Shear force measurements were conducted using a texture analyzer (TA.XT2, Stable Micro Systems Ltd., Surrey, UK). Tissue samples were prepared as 3 cm × 1 cm × 1 cm cubes and analyzed with a Warner-Bratzler blade under the following operational parameters: pre-test speed of 1 mm/s, mid-test speed of 3 mm/s, post-test speed of 5 mm/s, 50% deformation, and a 5 g trigger force.

2.7. Gut microbiota

Genomic DNA was extracted from rectal fecal samples (n = 7 pigs, and the other assays n = 8 differ because one intestinal content sample was lost during collection and was therefore unavailable for 16S rRNA sequencing) using a commercially available kit, and the integrity of the isolated DNA was verified via agarose gel electrophoresis. The PCR amplification was conducted using universal primers 338F (5′-ACTCCTACGGGAGGCAGCAG-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′). The resulting PCR products were quantified with the QuantiFluor-ST blue fluorescence quantification system (Promega Biotech Co., Ltd., Beijing, China). Sequencing was carried out on the Illumina HiSeq2500 PE 250 platform (Illumina Inc., San Diego, CA, USA). After performing quality control and filtering of the raw sequencing reads, FLASH software (version 1.2.11) was utilized to merge overlapping sequences and generate optimized data sets. The DADA2 algorithm was then applied for sequence denoising, classifying sequences with greater than 97% similarity into operational taxonomic units (OTUs). Using the representative sequences and their abundance data from the OTUs, various statistical and visualization analyses were performed, including taxonomic classification, community diversity assessment, differential species analysis, and correlation studies. Taxonomic annotation was based on the Silva138/16s_bacteria database, utilizing the classify sklearn (Naive Bayes) method with a classification confidence threshold of 0.7. Sequence denoising was performed with DADA2, quality control was executed using fastp (v0.19.6), and read merging was accomplished with FLASH (v1.2.7).

2.8. Statistical analysis

The pen was considered as the experimental unit for growth performance (n = 3), whereas all selected pigs from each pen were considered as the experimental unit for the other indexes. Data were expressed as means and standard error of the mean (SEM). Statistical evaluations were conducted using SPSS Statistics version 26 (IBM Analytics, Armonk, NY, USA). Except for using Student’s t-tests for fecal lipids, all other data were analyzed using one-way analysis of variance (ANOVA) and mixed linear model, followed by multiple comparisons employing the Dunnett test or Tamhane’s test. A P-value of less than 0.05 was considered statistically significant. The data were statistically analyzed using the mixed linear model in SPSS Statistics 26 software as follows:

Y_i = X_iβ + Z_iμ_i + ε_i

where Y_i is the dependent variable; X_iβ is the fixed part; Z_iμ_i is the random part; ε_i is the residual error. Orthogonal polynomial coefficients were used for linear or quadratic responses.

3. Results

3.1. Glycerol monolaurate improved growth performance

The effect of GML on growth performance of black pigs was first investigated. As shown in Table 2, compared with the NCD group, 750 mg/kg of GML significantly enhanced final body weight (linear, P = 0.008), ADG (linear, P = 0.016), and ADFI (linear, P = 0.007), but 250 mg/kg of GML had no significant influence on these indexes. Moreover, both 250 and 750 mg/kg of GML increased carcass yield and decreased backfat thickness in the trends (quadratic, P > 0.05).

Table 2.

The effects of glycerol monolaurate (GML) on growth performance.

Item	GML levels, mg/kg			SEM	P-value
	0	250	750		ANOVA	Linear	Quadratic
Initial body weight, kg	83.16	82.09	82.00	1.647	0.500	0.455	0.730
Final body weight, kg	106.83b	108.05ab	110.81a	2.008	0.031	0.008	0.459
ADG, g/d	422.82	463.53	514.38	48.714	0.068	0.016	0.857
ADFI, kg/d	2.63b	2.67b	2.78a	0.074	0.018	0.007	0.274
FCR	4.96	4.70	5.06	0.193	0.059	0.604	0.025
Carcass yield, %	71.64	72.28	72.41	0.337	0.569	0.317	0.708
Backfat thickness, cm	4.04	3.84	3.83	0.097	0.613	0.374	0.657

ADG = average daily weight gain; ADFI = average daily feed intake; FCR = feed conversion ratio; SEM = standard error of the mean.

Within a row, means without a common superscript letter differ at P < 0.05.

The repeats of initial body weight, final body weight, ADG, ADFI, and FCR was n = 3 pens/group. The repeats of carcass yield and backfat thickness was n = 6 pigs/group.

3.2. Glycerol monolaurate reduced fasting blood glucose and TG

To investigate the effects of GML on health status and glucolipid metabolism, liver and kidney function biomarkers and appetite hormones in the serum were measured. As shown in Table 3, compared with the NCD group, 250 and 750 mg/kg of GML markedly reduced fasting blood glucose (linear, P = 0.036) and TG (P = 0.044), and showed no significant influences on TC, LDL-C, HDL-C, and LDL-C/HDL-C ratio. The AKP, AST, and ALT are considered liver function biomarkers, and CRE and BUN are considered kidney function biomarkers. The three groups had no significant differences in the levels of AKP, AST, ALT, CRE, and BUN, indicating that obvious liver or kidney damages were not observed after dietary supplementation of 250 and 750 mg/kg of GML. To explore the reason for body weight gain and increased feed intake, the levels of appetite hormones were analyzed. Glucagon like peptide-1 and PYY belong to appetite suppressing hormones, while NPY and ghrelin belong to appetite promoting hormones. The three groups showed no significant differences in the levels of GLP-1, PYY, NPY, and ghrelin, except 750 mg/kg of GML significantly decreased ghrelin concentration (linear, P = 0.013), inferring that the body weight gain and increased feed intake were not ascribed to the regulation of appetite hormones. Moreover, the reduction of fasting blood glucose was not attributed to the regulation of GLP-1. Together, GML had no significant effects on serum biochemical indexes but reduced fasting blood glucose and TG.

Table 3.

The effects of glycerol monolaurate (GML) on serum biochemical indexes.

Item	GML levels, mg/kg			SEM	P-value
	0	250	750		ANOVA	Linear	Quadratic
Blood glucose, mmol/L	6.28a	4.63b	5.46b	0.676	0.033	0.036	0.117
TG, mmol/L	0.69a	0.45b	0.54ab	0.100	0.044	0.122	0.050
TC, mmol/L	3.41	3.47	3.29	0.070	0.826	0.680	0.645
HDL-C, mmol/L	1.26	1.48	1.36	0.089	0.548	0.632	0.328
LDL-C, mmol/L	1.71	1.71	1.71	0.003	0.999	0.992	0.958
LDL-C/HDL-C	1.26	1.48	1.36	0.089	0.548	0.632	0.328
AKP, U/L	41.90	36.06	46.21	4.158	0.172	0.538	0.080
AST, U/L	1.21	1.78	1.42	0.239	0.338	0.563	0.180
ALT, U/L	119.72	119.47	96.62	10.833	0.118	0.072	0.296
CRE, μmol/L	179.16	211.47	193.57	13.232	0.076	0.330	0.042
BUN, mmol/L	2.83	2.85	2.86	0.013	0.994	0.914	0.981
GLP-1, pg/mL	33.67	33.23	29.64	1.807	0.541	0.308	0.649
PYY, pg/mL	27.24	27.69	24.62	1.353	0.127	0.103	0.220
NPY, pg/mL	27.64	23.27	25.67	1.788	0.630	0.664	0.395
Ghrelin, pg/mL	221.92a	232.39a	161.27b	31.350	0.003	0.013	0.022

TG = total triglyceride; TC = total cholesterol; HDL-C = high-density lipoprotein cholesterol; LDL-C = low-density lipoprotein cholesterol; AKP = alkaline phosphatase; AST = aspartate aminotransferase; ALT = alanine aminotransferase; CRE = creatinine; BUN = blood urea nitrogen; GLP-1 = glucagon like peptide 1; PYY = peptide YY; NPY = neuropeptide Y; SEM = standard error of the mean.

Within a row, means without a common superscript letter differ at P < 0.05, n = 8 pigs/group.

3.3. Glycerol monolaurate decreased the concentration of LDL-C in the liver

To evaluate the effects of GML on liver health, the concentrations of TG, TC, HDL-C, and LDL-C in the liver were measured. As shown in Table 4, compared with the NCD group, 250, and 750 mg/kg of GML had no significant influence on TG, TC, and HDL-C levels, but significantly inhibited the LDL-C level (linear, P = 0.021), indicating that GML promoted the transport and metabolism of cholesterol in the liver.

Table 4.

The effects of glycerol monolaurate (GML) on hepatic lipid content (mmol/mg).

Item	GML levels, mg/kg			SEM	P-value
	0	250	750		ANOVA	Linear	Quadratic
TG	12.81	12.69	13.03	0.143	0.954	0.843	0.815
TC	6.05	6.16	6.55	0.212	0.478	0.243	0.703
HDL-C	1.09	0.82	0.70	0.162	0.798	0.499	0.890
LDL-C	5.65a	4.04b	3.92b	0.787	0.034	0.021	0.216

TG = total triglyceride; TC = total cholesterol; HDL-C = high-density lipoprotein cholesterol; LDL-C = low-density lipoprotein cholesterol; SEM = standard error of the mean.

Within a row, means without a common superscript letter differ at P < 0.05, n = 8 pigs/group.

3.4. Glycerol monolaurate increased the content of UFA in the liver

Fatty acid composition also reveals lipid metabolism in the liver. As shown in Table 5, a total of 17 fatty acids were detected in the liver, and the fatty acids with higher proportion in the three groups were stearic acid (C18:0), palmitic acid (C16:0), oleic acid (C18:1n9c), linoleic acid (C18:2n6c), and arachidonic acid (C20:4n6). Compared with the NCD group, 750 mg/kg of GML reduced the content of saturated fatty acid (SFA) and increased the content of UFA (P = 0.023) in the liver, among which monounsaturated fatty acid (MUFA; P = 0.009) was significantly increased but polyunsaturated fatty acid (PUFA; linear, P = 0.032) was significantly decreased. However, 250 mg/kg of GML had no similar effects. In terms of single fatty acid, 750 mg/kg of GML significantly decreased the content of C18:0 (linear, P = 0.007), C20:0 (P = 0.015), and significantly increased the content of C18:1n9c (P = 0.011) and C20:1 (P = 0.032). There were no significant differences in the content of all fatty acids between the NCD group and the GML250 group, except C20:1. Together, GML promoted the synthesis of UFA in the liver.

Table 5.

The effects of glycerol monolaurate (GML) on hepatic fatty acid composition.

Item	GML levels, mg/kg			SEM	P-value
	0	250	750		ANOVA	Linear	Quadratic
C16:0	14.53	14.77	15.89	0.592	0.107	0.047	0.434
C16:1	0.75	0.60	0.85	0.102	0.153	0.442	0.078
C17:0	0.57	0.61	0.51	0.042	0.674	0.591	0.481
C18:0	24.92a	24.80a	22.05b	1.326	0.008	0.007	0.112
C18:1n9c	13.72b	12.54b	17.62a	2.172	0.011	0.036	0.032
C18:2n6c	13.71	13.72	13.59	0.061	0.968	0.828	0.889
C18:3n6	0.22	0.18	0.24	0.024	0.219	0.575	0.103
C18:3n3	0.36	0.31	0.38	0.029	0.617	0.748	0.359
C20:0	0.18a	0.21a	0.09b	0.053	0.015	0.054	0.031
C20:1	0.17b	0.22a	0.22a	0.023	0.032	0.025	0.169
C20:2	0.53	0.55	0.58	0.022	0.382	0.165	0.818
C21:0	0.65	0.71	0.54	0.072	0.053	0.150	0.052
C20:3n6	0.59	0.61	0.52	0.037	0.603	0.442	0.515
C20:4n6	18.58	17.65	16.55	0.827	0.199	0.069	0.927
C22:0	0.38	0.38	0.39	0.005	0.956	0.813	0.854
C24:0	2.36	2.35	2.14	0.102	0.432	0.244	0.544
C24:1	0.87	0.68	0.78	0.076	0.232	0.436	0.132
SFA	43.59	43.83	41.60	0.997	0.097	0.082	0.196
MUFA	15.51b	14.04b	19.47a	2.295	0.009	0.040	0.022
PUFA	33.98	33.02	31.86	0.867	0.107	0.032	0.910
UFA	49.49ab	47.05b	51.33a	1.753	0.023	0.020	0.013

SFA = saturated fatty acid; UFA = unsaturated fatty acid; MUFA = monounsaturated fatty acid; PUFA = polyunsaturated fatty acid; SEM = standard error of the mean.

Within a row, means without a common superscript letter differ at P < 0.05, n = 8 pigs/group.

3.5. Glycerol monolaurate improved meat quality

The effects of GML on meat quality of muscle tissue are shown in Table 6. The meat color includes lightness (L∗), redness (a∗), and yellowness (b∗). Compared with the NCD group, 250 and 750 mg/kg of GML increased the a∗ value by 4.95% and 14.68%, respectively, but had no significant influence. The lower post-mortem pH value indicates a faster glycolysis rate, and the smaller drip loss represents better water-holding capacity. The pH value of 250 and 750 mg/kg of GML increased by 1.53% and 5.78%, respectively, and 750 mg/kg of GML reduced drip loss by 7.94% but 250 mg/kg of GML increased drip loss by 8.36%, indicating that 750 mg/kg of GML induced a better meat quality. The smaller shear force represents the bigger tenderness. GML at 750 mg/kg prominently decreased the shear force of muscle tissue (P = 0.002), showing that 750 mg/kg of GML strengthens tenderness. Nutritional composition of pork mainly includes moisture content, CP, crude fat, and ash content. GML at 750 mg/kg decreased moisture content in the trend (P = 0.109) and significantly increased CP content (P = 0.034), but no obvious influences were observed in ash content and crude fat content compared with the NCD group, which suggested that GML promoted protein deposition. In brief, GML supplementation improved the meat quality in black pigs.

Table 6.

The effects of glycerol monolaurate (GML) on meat quality.

Item	GML levels, mg/kg			SEM	P-value
	0	250	750		ANOVA	Linear	Quadratic
Lightness (L∗)	42.46	42.16	41.24	0.520	0.934	0.719	0.920
Redness (a∗)	3.37	3.52	3.86	0.207	0.680	0.385	0.841
Yellowness (b∗)	7.40	7.22	7.59	0.150	0.815	0.738	0.590
pH24 h	5.41	5.50	5.73	0.133	0.197	0.077	0.629
Drip loss, %	2.73	2.96	2.51	0.182	0.598	0.621	0.382
Shear force, g	1253.89a	1367.60a	1036.42b	137.397	0.002	0.043	0.005
Moisture content, %	72.86	73.32	71.75	0.659	0.109	0.155	0.122
CP, %	21.46b	21.26b	22.39a	0.489	0.034	0.051	0.083
Crude fat, %	3.09	2.94	3.07	0.067	0.974	0.986	0.823
Ash content, %	1.23	1.18	1.17	0.029	0.119	0.056	0.396

CP = crude protein; SEM = standard error of the mean.

Within a row, means without a common superscript letter differ at P < 0.05, n = 6 pigs/group.

3.6. Glycerol monolaurate promoted AA synthesis

Amino acid composition relates to the nutritional function of pork and also participates in flavor formation. To further reveal the difference in protein content, AA composition was analyzed. As shown in Table 7, a total of 17 kinds of AA were detected. Compared with the NCD group, 750 mg/kg of GML increased the contents of total AA, essential AA, non-essential AA, umami AA, and sweet AA (P < 0.05), but 250 mg/kg of GML markedly decreased the contents of these AA (P < 0.05). In detail, 750 mg/kg of GML increased the content of every AA, in which the contents of Ala, Met, Leu, Tyr, Lys, His, and Arg were significantly increased (P < 0.05). However, most of the AA in the GML 250 group were significantly decreased. These results suggested GML increased protein content of muscle tissues by promoting AA synthesis.

Table 7.

The effects of glycerol monolaurate (GML) on amino acid (AA) composition of muscle tissues (mg/g).

Item		GML levels, mg/kg			SEM	P-value
		0	250	750		ANOVA	Linear	Quadratic
1	Asp	1.26a	1.07b	1.34a	0.114	0.011	0.423	0.004
2	Thr	0.55ab	0.45b	0.59a	0.062	0.006	0.475	0.002
3	Ser	0.41a	0.32b	0.43a	0.051	0.006	0.683	0.002
4	Glu	2.37ab	2.01b	2.53a	0.217	0.027	0.559	0.010
5	Ala	0.61b	0.48c	0.71a	0.092	0.001	0.141	<0.001
6	Gly	0.62a	0.51b	0.70a	0.010	0.001	0.206	<0.001
7	Val	0.05	0.05	0.07	0.010	0.062	0.093	0.099
8	Cys	1.43ab	1.18b	1.65a	0.193	0.001	0.139	0.001
9	Met	0.35b	0.27c	0.42a	0.059	<0.001	0.105	<0.001
10	Ile	0.64ab	0.52b	0.72a	0.085	0.001	0.193	0.001
11	Leu	1.05b	0.84c	1.22a	0.153	<0.001	0.130	<0.001
12	Phe	0.40ab	0.32b	0.46a	0.060	0.001	0.155	<0.001
13	Tyr	0.58b	0.47c	0.66a	0.081	<0.001	0.159	<0.001
14	Lys	1.22b	0.97c	1.41a	0.178	<0.001	0.148	<0.001
15	His	1.90b	1.63c	2.21a	0.235	<0.001	0.070	<0.001
16	Arg	0.82ab	0.64b	0.98a	0.138	<0.001	0.108	<0.001
17	Pro	0.56	0.52	0.46	0.038	0.474	0.223	0.813
Total AA		14.82ab	12.25b	16.31a	1.675	0.005	0.387	0.002
Essential AA		4.26b	3.42c	4.90a	0.605	<0.001	0.162	<0.001
Non-essential AA		10.56a	8.83b	11.41a	1.073	0.009	0.432	0.003
Umami AA		3.63ab	3.08b	3.85a	0.322	0.025	0.599	0.009
Sweet AA		2.75ab	2.28b	2.89a	0.264	0.034	0.592	0.012

SEM = standard error of the mean.

Within a row, means without a common superscript letter differ at P < 0.05, n = 6 pigs/group.

3.7. Glycerol monolaurate had no significant effect on fatty acid composition of muscle tissue

The effect of GML on fatty acid composition of muscle tissues is shown in Table 8. No significant differences in fatty acid composition were observed among the three groups (P > 0.05), except GML significantly decreased the content of C20:1 (P = 0.013).

Table 8.

The effects of glycerol monolaurate (GML) on fatty acid composition of muscle tissues (%).

Item	GML levels, mg/kg			SEM	P-value
	0	250	750		ANOVA	Linear	Quadratic
C4:0	0.07	0.09	0.07	0.007	0.558	0.879	0.293
C6:0	0.02	0.02	0.02	0.001	0.770	0.862	0.490
C8:0	0.01	0.02	0.01	0.001	0.472	0.440	0.344
C10:0	0.08b	0.10a	0.08b	0.006	0.030	0.869	0.009
C12:0	0.12	0.13	0.13	0.001	0.986	0.902	0.908
C13:0	0.02	0.02	0.02	0.002	0.557	0.990	0.288
C14:0	1.14	1.21	1.08	0.050	0.527	0.629	0.313
C14:1	0.02	0.02	0.02	0.002	0.524	0.821	0.273
C15:0	0.02	0.02	0.02	0.002	0.845	0.724	0.648
C15:1	0.92	1.07	0.87	0.085	0.606	0.842	0.335
C16:0	24.20	24.00	23.73	0.196	0.854	0.569	0.958
C16:1	2.90	3.00	2.99	0.047	0.951	0.795	0.852
C17:1	0.43	0.48	0.47	0.025	0.836	0.670	0.676
C18:0	12.30	12.34	12.16	0.075	0.949	0.806	0.831
C18:1n9c	45.24	44.35	46.04	0.693	0.297	0.465	0.174
C18:2n6c	8.57	9.09	8.61	0.237	0.865	0.968	0.597
C18:3n6	0.06	0.05	0.04	0.005	0.533	0.271	0.772
C18:3n3	0.33	0.31	0.30	0.013	0.675	0.367	0.991
C20:0	0.25	0.24	0.20	0.020	0.109	0.047	0.437
C20:1	0.74a	0.59b	0.59b	0.070	0.013	0.011	0.127
C20:2	0.40	0.38	0.36	0.019	0.618	0.320	0.959
C21:0	0.23	0.24	0.22	0.008	0.920	0.750	0.797
C20:3n6	1.39	1.61	1.47	0.093	0.780	0.790	0.519
C20:4n6	0.14	0.13	0.12	0.011	0.458	0.207	0.890
C20:3n3	0.03	0.04	0.03	0.004	0.497	0.753	0.262
C22:0	0.10	0.12	0.09	0.009	0.392	0.655	0.202
C20:5	0.04	0.05	0.02	0.014	0.106	0.329	0.063
C23:0	0.03	0.02	0.02	0.003	0.118	0.049	0.480
C24:0	0.15	0.19	0.15	0.016	0.537	0.926	0.274
C24:1	0.07	0.08	0.07	0.006	0.623	0.901	0.343
ω-6	10.15	10.88	10.24	0.325	0.841	0.947	0.565
ω-3	0.40	0.41	0.35	0.024	0.445	0.327	0.414
SFA	38.75	38.74	38.01	0.347	0.774	0.525	0.731
MUFA	50.30	49.60	51.05	0.591	0.462	0.522	0.292
PUFA	10.95	11.66	10.95	0.338	0.845	0.997	0.569
UFA	61.25	61.26	61.99	0.347	0.774	0.525	0.731
SFA/UFA	0.64	0.63	0.61	0.009	0.768	0.503	0.763

SFA = saturated fatty acids; UFA = unsaturated fatty acids; MUFA = monounsaturated fatty acids; PUFA = polyunsaturated fatty acids; SEM = standard error of the mean.

Within a row, means without a common superscript letter differ at P < 0.05, n = 6 pigs/group.

3.8. Glycerol monolaurate regulated gut microbiota

Gut microbiota is closely related to health status and meat quality of farming animals. The α-diversity reflects richness and diversity of gut microbiota, including abundance-based coverage estimator‌ (ACE), Chao, Shannon, and Simpson index. As shown in Fig. 1A–D, there were no prominent differences in the four indexes among the three groups, meaning that GML showed no obvious changes in the richness and diversity of gut microbiota. The result of principal coordinate analysis (PCoA) analysis also confirmed this conclusion (Fig. 1F). There were shared 202 OTUs in three groups, and special OTUs of the NCD group, GML250 group, and GML750 group were 19, 46, and 47, respectively (Fig. 1E). Then, the gut microbiota composition at the phylum level was analyzed, and found that Firmicutes and Bacteroidota were the dominant bacteria with a proportion over 90% (Fig. 1G), followed by Actinobacteriota, Spirochaetota, and Proteobacteria. At the genus level, the top 10 of relative abundance were Terrisporobacter, Romboutsia, Prevotellaceae, Eggerthellaceae, Parabacteroides, Eubacterium_hallii_group, and so on (Fig. 1H). Compared with the NCD group, 250 mg/kg of GML significantly increased the relative abundances of Lachnospiraceae_AC2044_group and Parabacteroides (Fig. 1I), but 750 mg/kg of GML significantly increased the relative abundances of Terrisporobacter, Romboutsia, Eggerthellaceae, and Parabacteroides (Fig. 1J). Meanwhile, compared with the GML250 group and 750 mg/kg of GML significantly increased the relative abundances of Terrisporobacter, Romboutsia, Turicibacter, Marvinbryantia, norank_f_Ruminococcaceae, Lachnospiraceae_FCS020_group, Oscillospira, Lachnospiraceae_NK3A20_group, Oscillibacter, Fibrobacter (Fig. 1K). Together, the Terrisporobacter and Romboutsia could be considered characteristic bacteria to distinguish differences in gut microbiota composition between groups.

Fig. 1.

The effects of glycerol monolaurate (GML) on gut microbiota. (A) Abundance-based coverage estimator (ACE) index. (B) Chao index. (C) Shannon index. (D) Simpson index. (E) Venn diagram. (F) Principal coordinate analysis (PCoA). (G) Gut microbiota composition at the phylum level. (H) Gut microbiota composition at the genus level. (I) The comparison of gut microbiota composition between the NCD and GML250 groups at the genus level. (J) The comparison of gut microbiota composition between the NCD and GML750 groups at the genus level. (K) The comparison of gut microbiota composition between the GML250 and GML750 groups at the genus level. NCD, control group, basal diet; GML250 group, basal diet with 250 mg/kg of GML group; GML750 group, basal diet with 750 mg/kg of GML. ∗ Indicates a statistically significant difference between groups at P < 0.05, ∗∗ indicates a higher statistically significant difference between groups at P < 0.01; n = 7 pigs/group.

3.9. Glycerol monolaurate increased TBA content in the feces

It was affected the content of fecal lipids in view of GML as an emulsifier was investigated. Based on previous results, the GML750 group showed a better regulatory effect than the GML250 group, so the comparisons between NCD group and the GML750 group were only analyzed. As shown in Table 9, no significant differences in the content of fecal lipids, fecal TG and fecal TC were observed between the two groups (P > 0.05), but 750 mg/kg of GML significantly increased fecal TBA content (P = 0.001), indicating that GML promoted the secretion of bile acid in the feces.

Table 9.

The effects of glycerol monolaurate (GML) on fecal lipids.

Item	GML levels, mg/kg		SEM	P-value
	0	750
Total lipid, %	7.43	6.64	0.396	0.162
TG, mmol/mg	1.19	1.18	0.006	0.951
TC, mmol/mg	1.51	1.58	0.039	0.586
TBA, μmol/L	205.93b	262.84a	28.455	0.001

TG = total triglyceride; TC = total cholesterol; TBA = total bile acid; SEM = standard error of the mean.

Within a row, means without a common superscript letter differ at P < 0.01, n = 8 pigs/group.

4. Discussion

The Duroc × Landrace × Yorkshire crossbred pigs have the largest market share in China, and are highly favored by farmers due to their rapid growth rate, low FCR, and good economic gain. However, their production is quantity driven, often resulting in inferior pork quality (Huang et al., 2020). In contrast, Chinese indigenous pigs are valued for their superior meat quality. However, Chinese indigenous pigs have a slower growth rate and higher production costs (Tu et al., 2021). Nutrition regulation strategy is an effective and safe method to improve growth performance and meat quality of Chinese indigenous pigs, such as the use of organic acids, minerals, and prebiotics (Liu et al., 2018; Weng et al., 2025). In this study, dietary GML supplementation significantly increased final body weight, ADG, and ADFI of finishing pigs, indicating improved growth performance and shortened production cycles. Importantly, body weight gain was not attributed to obesity or increased appetite, as GML sharply reduced fasting blood glucose and serum TG without affecting appetite hormones, TC, HDL-C, or LDL-C in the serum. The conclusion that GML reduced fasting blood glucose and serum TG has also been found in other studies, which confirmed that GML performed more pronounced regulation than lauric acid and lauric triglyceride on glucose homeostasis and lipid metabolism in high-fat diet-fed mice and zebrafish (Wang et al., 2022; Zhao et al., 2022). The results of this study also provided new evidence for the hypoglycemic and lipid-lowering effects of GML. Moreover, the concentrations of appetite hormones in the serum were not increased after GML supplementation, so body weight gain might be ascribed to the fact that GML promoted the metabolism and absorption of dietary nutrients by increasing digestive enzyme activities or altering intestinal morphology and mucosal immunity (Wang et al., 2021, Wang et al., 2021; Zhao et al., 2023).

Disruption of hepatic lipid metabolism leads to hepatic inflammation, hepatocellular damage, and lipotoxicity-induced hepatic injury (Geng et al., 2021). In this study, GML decreased the content of SFA and increased the content of UFA in the liver, which might be because GML modulated glycerophospholipid metabolism and promoted β-oxidation of fatty acids (Zhao et al., 2020), resulting in the transport of fatty acids and other lipids from the liver to other tissues for energy consumption. This inference could also be supported by the result of hepatic LDL-C, because LDL-C as a lipid transporter has been widely recognized. On the other hand, GML may enhance UFA biosynthesis in the liver, potentially increasing MUFA while decreasing SFA. This shift likely stems from upregulated expression of stearoyl-CoA desaturase and fatty acid desaturase genes (Chen et al., 2022). For health reasons, the consumer increasingly prefers products with a higher UFA content, and previous studies reported that PUFA were mainly applied to increase the concentration of UFA in pig tissues, such as linolenic acid or conjugated linoleic acid (Rossi et al., 2010; Wang et al., 2021, Wang et al., 2021). However, pig diets supplemented with PUFA are more easily oxidized and cause more storage problems. Glycerol monolaurate could be an alternative strategy based on its high oxidative stability and promotion of unsaturated fatty acid synthesis.

Meat quality broadly includes sensory characteristics, nutritional characteristics, and safety characteristics, and they are not only inherent properties of meat products, but also key points for consumers to purchase and make decisions (Warner et al., 2010). Sensory characteristics are the most direct and effective evaluation method, and intuitive perception directly reflects the quality of pork, such as meat color, pH value, drip loss, and shear force. In this study, GML evidently increased meat redness and pH value, decreased drip loss and shear force, showing that GML improved meat quality. The decrease in shear force means an increase in tenderness, and this effect might be because GML increased muscle fiber density and reduced muscle fiber diameter (Zhuang et al., 2022), resulting in muscle fibers being more prone to fracture when subjected to shear stress. In terms of nutritional characteristics, GML significantly increased CP content of skeletal muscle, but had no prominent influences on crude lipid, moisture, ash, and fatty acid composition. Combined with the result of AA composition, GML increased the content of every AA (except Val and Pro), meaning that GML increased CP content by promoting the synthesis and deposition of AA. A similar conclusion was also confirmed in our previous study, in which medium-chain monoglycerides significantly promoted the five pathways related to AA metabolism involving alanine metabolism, aspartate and glutamate metabolism, glycine, serine and threonine metabolism, histidine metabolism, arginine and proline metabolism in fresh meat of broilers (Liu et al., 2021a, Liu et al., 2021b). The increase in AA and CP partly explain the body weight gain and increased carcass yield, meaning that dietary nutrients might be deposited or transformed in the skeletal muscle in the form of proteins. Future researches need to focus on exploring the mechanism of GML on AA synthesis.

The intestines of mammals are filled with a large number of gut microbes, which coordinate with the hosts to achieve a stable gut micro-environment (Ma et al., 2022). As the indigenous inhabitants of the gut, the gut microbiota assists the host in digestion and absorption of dietary components, and also produces secondary metabolites to regulate gut function and communicate or interact with distant tissues and organs, such as short chain fatty acids, bile acids, and trimethylamine (Nogal et al., 2021). As a well-known antibacterial agent and emulsifier, the regulatory effect of GML on gut microbiota has been widely studied (Luo et al., 2022). Our previous studies also found that GML had significant effects on the growth and meat quality of farmed animals, which was closely related to the gut microbiota (Liu et al., 2020a; Liu et al., 2020b; Wang et al., 2021, Wang et al., 2021). In this study, GML supplementation had no noteworthy influence on the richness and diversity of gut microbiota, but significantly increased the relative abundances of Terrisporobacter and Romboutsia compared with the NCD and GML250 groups. As an anaerobic gram-positive bacterium, the increased abundance of Terrisporobacter was related to the increased concentration of partial secondary bile acids in weaned pigs (Adekolurejo et al., 2023), which might be because the enzyme activity of bile salt hydrolase derived from Terrisporobacter was changed (Liu et al., 2023). Romboutsia also had the activity of bile acid-hydrolysis, and an increase of its relative abundance might be accompanied by an increase of secondary bile acids, which was supported by a previous study (Xie et al., 2023). These studies suggested that increased abundances of Terrisporobacter and Romboutsia after GML supplementation might result in an increase in bile acids in the feces. This hypothesis was supported by the result of TBA in the feces, which meant that bile acids played an important role. Bile acids are cholesterol metabolites to facilitate the absorption and excretion of dietary lipids, and are considered signaling molecules to modulate glucolipid metabolism and immune homeostasis (Collins et al., 2023). The increase of fecal bile acids could also lead to the excretion of hepatic cholesterol, which was beneficial in reducing the content of LDL-C in the liver. It was consistent with the result of hepatic LDL-C. Given their multiple beneficial effects on farmed animals, bile acids have been approved as feed additives in China since 2014 (MOA, 2014). In addition to the growth promoting effect, bile acids reduce blood glucose and triglyceride in the serum (Yang et al., 2022; Zheng et al., 2021), which parallels the reductions in blood glucose and triglyceride in this study. However, whether these effects were directly mediated by bile acids remain unclear. Furthermore, the contribution of bile acids to meat quality improvements in black pigs is unknown, because bile acids mainly promote the absorption and digestion of dietary lipids, instead of protein or AA synthesis. Another study found that bile acids had no apparent effects on the meat quality in lamb (Zhang et al., 2022). Current evidences indicate that GML has primarily been supplemented in sows or weaned piglets, with research focusing on its roles in improving intestinal health and alleviating inflammation. However, its application in finishing pigs has received scant attention, especially in terms of growth performance and meat quality (Zhao et al., 2023; Xiong et al., 2025). Together, this study first confirmed that GML promoted growth performance and improved meat quality of finishing pigs, and these effects were closely related to gut microbiota and bile acids. However, whether gut microbiota or bile acids played the key role in these changes is unknown, and warrants further investigation using single strain transplantation or bile acid supplementation experiments.

5. Conclusion

In this study, GML supplementation promoted body weight gain and feed intake of black pigs, without causing disturbances in glucose and lipid metabolism. GML supplementation also improved meat quality, increased protein deposition in muscle and promoted the production of UFA in the liver. These effects might be attributed to the changes in gut microbiota composition and fecal bile acid profiles. Overall, this study provides basic data supporting the application of GML as a functional feed additive to improve growth performance and meat quality in finishing pigs and potentially other farm animals.

Credit Author Statement

Nanhai Xiao: Writing – original draft, Software, Project administration, Methodology, Investigation. Xutang Wang: Resources, Methodology, Investigation. Jing Wang: Formal analysis, Data curation. Haiying Cai: Visualization, Validation. Fengqin Feng: Writing – review & editing, Supervision, Conceptualization. Minjie Zhao: Writing – review & editing, Supervision.

Declaration of competing interest

We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, and there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the content of this paper.