A 12-week randomized, double-blind, placebo-controlled exploratory pilot study of HFN2-008 (heat-treated Bifidobacterium longum HN001) for adiposity reduction in overweight adults

Abstract

Background

Excessive accumulation of body fat, particularly abdominal and visceral fat, is a major risk factor for metabolic abnormalities and chronic diseases. This study evaluated the safety and effects of 12-week HFN2-008 supplementation on body fat distribution and abdominal fat in adults.

Methods

A 12-week randomized, double-blind, placebo-controlled exploratory pilot study was conducted in 100 adult men and women. Participants were randomly assigned to receive either HFN2-008, a formulation containing heat-treated Bifidobacterium longum HN001 dried culture powder, or a placebo. Body fat mass and percentage were assessed using dual-energy X-ray absorptiometry, whereas abdominal fat areas were assessed using computed tomography.

Results

After 12 weeks of supplementation, the HFN2-008 group demonstrated significantly greater decreases in trunk and total body fat mass and percentage compared with the placebo group. Computed tomography further revealed significant decreases in visceral and subcutaneous fat areas in the HFN2-008 group compared with the placebo group. No significant between-group differences were observed in lean body mass or circulating metabolic biomarkers. No serious adverse events were reported, indicating a favorable safety profile.

Conclusions

HFN2-008 effectively decreases body and abdominal fat without loss of lean body mass and appears to be a safe functional ingredient for body fat management in adults.

Trial registration

Registry: Clinical Research Information Service, TRN: KCT0011178, Registration date: 19 November 2025

Introduction

Rapid economic growth and industrialization have substantially altered lifestyles and dietary patterns, thereby increasing the prevalence of obesity globally. Diets characterized by high fat and sugar intake, together with low physical activity and lower energy expenditure, create conditions that promote excessive fat accumulation. Additionally, irregular eating habits and frequent consumption of high-calorie snacks and late-night meals further increase the risk of obesity [1].

Obesity is defined as a condition of excessive fat accumulation resulting from an imbalance between energy intake and expenditure. It is recognized as a major risk factor for metabolic abnormalities, including impaired immune function, hypertension, type 2 diabetes mellitus, and dyslipidemia. Long-term obesity significantly increases the risk of chronic diseases, such as cardiovascular and cerebrovascular disorders, thereby increasing morbidity and mortality rates [2, 3].

According to the World Health Organization, approximately 39% of adults aged 18 years and older worldwide were classified as overweight in 2016, with 13% classified as obese. Obesity has been designated as one of the most critical chronic diseases of the 21st century. In Korea, the prevalence of adult obesity continues to rise, representing a major public health concern that imposes substantial social and economic burdens [4, 5].

Currently, interventions related to diet and exercise are the primary strategies for obesity management, and pharmacological or surgical interventions are only applied when necessary. However, these approaches are often limited by poor long-term adherence and potential adverse effects. Consequently, there is an increasing demand for safer and more sustainable approaches to body fat management. In this context, functional foods that can suppress fat accumulation or modulate energy metabolism while minimizing adverse effects have gained attention as alternative approaches [6].

Recent evidence has highlighted the important role of gut microbiota in regulating obesity and energy metabolism. Alterations in the composition of gut microbiota are closely associated with host energy harvest, fat accumulation, and inflammatory responses. For instance, recent systematic reviews and meta-analyses of human clinical studies have demonstrated that modulating the gut microbiota through probiotic or postbiotic interventions can significantly reduce body weight, adiposity, and metabolic abnormalities [7].

In addition to probiotics, postbiotics are gaining attention, including heat-treated bacterial cells and dried culture preparations. Postbiotics comprise microbe-derived metabolites and structural components, including short-chain fatty acids, polysaccharides, bacteriocins, and cell wall constituents, and are characterized by enhanced storage stability and safety. Although accumulating evidence suggests that postbiotics exert anti-inflammatory, immunomodulatory, anti-obesity, and lipid metabolism-improving effects, clinical evidence from human studies evaluating their effects on body fat loss remains limited [8, 9].

Bifidobacterium species are nonmotile, obligate anaerobic, Gram-positive bacteria of the phylum Actinobacteria and, along with Lactobacillus species, represent one of the most extensively studied groups of intestinal probiotics. Among them, Bifidobacterium longum is abundantly present in the human gastrointestinal tract and contributes to host energy metabolism and immune regulation by producing bioactive compounds, such as short-chain fatty acids, acetate, and lactate [10]. Owing to these health benefits, B. longum has been widely utilized in the development of probiotic-based functional foods [11]. Recent studies using animal models with induced obesity have demonstrated that heat-treated B. longum-derived paraprobiotics or dried culture preparations exert beneficial effects on decreasing body weight and fat mass [12, 13]. However, randomized, double-blind, placebo-controlled, human clinical studies evaluating the efficacy and safety of formulations containing heat-treated B. longum HN001 dried culture powder remain limited.

The present study was designed as a randomized, double-blind, placebo-controlled, human intervention study and evaluated the safety and effects of 12-week supplementation with HFN2-008, a formulation containing heat-treated B. longum HN001 dried culture powder, on body fat loss and abdominal fat distribution in adults. We hypothesized that daily supplementation with HFN2-008 (500 mg/day) would safely and effectively reduce body fat in overweight adults. The 12-week intervention duration was selected as it is considered an adequate timeframe to observe meaningful changes in body composition and metabolic parameters based on previous clinical studies. To test this hypothesis, the primary outcomes evaluated were the reductions in total body fat mass, body fat percentage, and abdominal fat areas (total, visceral, and subcutaneous).

Materials and methods

Study participants

This study was conducted from January 22, 2025 (first participant screening), to August 18, 2025 (final visit of the last participant). This human intervention study was conducted at H+ Yangji Hospital, Seoul Hyocheon Medical Foundation (Seoul, Republic of Korea), in accordance with the principles of the Declaration of Helsinki and Good Clinical Practice guidelines. The study was reported in accordance with the CONSORT guidelines and approved by the Institutional Review Board of H+ Yangji Hospital (approval no. 2024-11-007). All participants received a full explanation of the study objectives and procedures and provided written informed consent prior to participation. The primary and secondary outcomes were predefined in the study protocol before participant enrollment. Although the study was not prospectively registered, it was retrospectively registered at the Clinical Research Information Service (CRIS; TRN: KCT0011178, Registration date: 19 November 2025). This retrospective registration resulted from an oversight in ensuring prospective registration prior to participant enrollment. However, the protocol was registered strictly before data lock, unblinding, and statistical analysis, ensuring that all outcomes were evaluated according to the pre-specified protocol without any post-hoc modifications.

Eligible male and female participants were aged 19–65 years with a body mass index (BMI) of 25.0–29.9 kg/m², classified as overweight, and were not taking medications known to affect body weight or metabolism during the study period. Participants were required to understand the purpose and procedures of the study and demonstrate the ability to comply with all study requirements.

Exclusion criteria included body weight changes of ≥ ± 5 kg within the past 3 months, surgery within the past 6 months, or bariatric surgery within the past year. The following exclusion criteria were also applied: psychiatric disorders or a history of psychological treatment, cardiovascular or cerebrovascular disease, uncontrolled diabetes mellitus, severe gastrointestinal disease, hepatic or renal failure, malabsorption syndromes, autoimmune diseases, cancer, thyroid disorders, pregnant or lactating women, women planning pregnancy during the study period, women who had given birth within the past 6 months, anti-obesity drugs, antidiabetic medications, steroids, hormone therapies, excessive alcohol consumption or smoking habits, participation in weight loss programs, and consumption of functional foods or probiotic supplements. Participants with abnormal findings in clinical laboratory tests and those deemed unsuitable for participation by the principal investigator were also excluded.

The sample size for this study was determined based on previously reported mean changes in body fat mass, which was considered the main efficacy parameter for planning purposes, over a 12-week intervention period [14]. Assuming a two-sided significance level of 0.05 and a statistical power of 80%, a minimum of 43 participants per group was required to detect a difference in mean changes between the two groups. Considering an anticipated dropout rate of 15%, the target sample size was set at 50 participants per group, for a total of 100 participants.

Study design

This study was designed as a randomized, double-blind, placebo-controlled exploratory pilot study. Eligibility was assessed during the screening visit based on predefined inclusion and exclusion criteria. Eligible participants at the baseline visit were randomly assigned at a 1:1 ratio to either the HFN2-008 group or placebo group. The randomization sequence was generated by an independent statistician utilizing SAS^® software (version 9.4; SAS Institute, Cary, NC, USA). A block randomization method was applied to ensure an equal allocation between the two groups.

Participants in the HFN2-008 group received the test product, HFN2-008, whereas those in the placebo group received a placebo identical in appearance. HFN2-008 was a capsule-type product containing heat-treated, dried B. longum HN001 as the main ingredient and was manufactured by Huons-N Co., Ltd. (Republic of Korea). The thermal inactivation process was performed at 100 °C for 30 min. The test formulation consisted of 12.5% B. longum HN001 powder and 87.5% excipients (86.5% corn starch and 1.0% magnesium stearate). The placebo was manufactured to be completely identical in appearance (including shape and color), flavor, weight, and packaging to achieve a rigorous double-blind design. Specifically, the complete formulation of the placebo consisted of 99.0% corn starch and 1.0% magnesium stearate.

All randomized participants consumed either HFN2-008 or placebo for 12 weeks, taking one capsule twice daily with sufficient water after meals, delivering a total daily dose of 500 mg of HFN2-008 (providing 1.25 × 10¹⁰ cells/day). During the 12-week intervention period, participants were instructed to maintain their usual dietary habits and physical activity levels, and were asked not to initiate any new weight-loss programs. The randomization codes were securely sealed and strictly maintained, remaining unopened until the completion of the study and final database lock prior to statistical analysis. Participants visited the study site according to a predefined schedule, during which their safety and compliance were systematically monitored. Compliance was assessed at each visit by counting the returned capsules.

Outcome measures and assessments

Demographic characteristics were analyzed in the Safety Set, defined as all participants who consumed the study product at least once. Collected variables included sex, age, height, weight, alcohol consumption, and smoking status, and were used to assess baseline homogeneity between the two groups.

Efficacy analyses were performed in the Per Protocol Set, which included participants who completed the study without major protocol violations. Given the exploratory and hypothesis-generating nature of this pilot study, efficacy outcomes focused on compliant participants (Per Protocol Set) to identify potential efficacy signals, rather than strictly performing an intention-to-treat (ITT) analysis for confirmatory testing. Data deemed clinically implausible or indicative of measurement errors were excluded from the analysis. Efficacy endpoints were classified as primary or secondary outcomes.

The primary efficacy outcomes focused on indicators related to body fat loss. Total body fat mass and percentage were measured at baseline and after 12 weeks using dual-energy X-ray absorptiometry (DEXA). Additionally, total abdominal, visceral, and subcutaneous fat area and the visceral-to-subcutaneous fat area ratio were assessed using computed tomography (CT). Abdominal fat area was analyzed from a single-slice CT image obtained at the level of the fourth to fifth lumbar vertebrae (L4-L5). To ensure accuracy and minimize variations, both DEXA and CT measurements were performed in the morning after an overnight fast of at least 12 h. Participants were instructed to wear standard light clothing and to remove all metallic objects prior to the scans.

Secondary efficacy outcomes included body composition, anthropometric parameters, and metabolic biomarkers. Lean body mass was assessed using DEXA. Anthropometric measurements included body weight, BMI, waist circumference, hip circumference, and the waist-to-hip ratio. Waist circumference was systematically measured at the midpoint between the lower margin of the last palpable rib and the top of the iliac crest, using a non-stretchable measuring tape at the end of normal expiration. Blood samples were analyzed for lipid-related parameters, high-sensitivity C-reactive protein, leptin, insulin, and adiponectin. Additionally, uncoupling protein 1 (UCP1) and peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α), which are involved in regulating energy metabolism, were evaluated.

Safety assessments were conducted in the Safety Set and included documenting all adverse events occurring during the study period. Overall safety was evaluated through clinical laboratory tests, vital signs, electrocardiography, and physical examinations.

Statistical analysis

All statistical analyses were performed using SAS software (version 9.4; SAS Institute, Cary, NC, USA) and SPSS software (version 26.0; IBM Corp., Armonk, NY, USA). Statistical significance was defined as a two-sided p-value of < 0.05. Demographic characteristics were analyzed using descriptive statistics. Continuous variables are presented as mean ± standard deviation, and categorical variables are expressed as frequencies and percentages.

Efficacy analyses were primarily conducted using the Per Protocol Set. Changes from baseline were calculated for each outcome variable by group. Between-group comparisons were performed using independent t-tests or Wilcoxon rank-sum tests, depending on the normality of data distribution. Within-group changes were analyzed using paired t-tests or Wilcoxon signed-rank tests. When significant between-group differences were observed, analysis of covariance (ANCOVA) was additionally performed with baseline values included as covariates. Safety analyses were conducted in the Safety Set. The incidences of adverse events are presented as frequencies and proportions by group, and between-group comparisons were performed using the chi-square test or Fisher’s exact test, as appropriate. Given the exploratory nature of this pilot study, which aimed to generate hypotheses across multiple adiposity parameters rather than strictly test a single confirmatory hypothesis, specific statistical adjustments for multiple comparisons were not applied. Thus, the presented p-values should be interpreted as exploratory.

Results

Demographic characteristics and participant flow

A total of 140 individuals were screened for eligibility, of which 40 were excluded due to exclusion criteria or withdrawal of informed consent. As such, 100 participants were randomized, of which five did not consume the study product at least once and were thus excluded, and 95 participants were included in the Safety Set. After excluding participants who discontinued the study, demonstrated insufficient compliance, or had major protocol violations, a total of 86 participants (42 in the HFN2-008 group and 44 in the placebo group) were included in the Per Protocol Set for the primary efficacy analysis (Fig. 1).

Fig. 1.

Flow diagram of participant recruitment, randomization, and analysis sets in the study assessing the efficacy and safety of HFN2-008. Abbreviations: IP, investigational product; ITT, intention-to-treat; PP, per-protocol

At baseline, no significant differences (p > 0.05) between the two groups were observed regarding demographic characteristics, including sex distribution, age, height, body weight, BMI, alcohol consumption, and smoking status. All demographic variables were evenly distributed between the two groups, confirming baseline similarity (Table 1).

Table 1.

Baseline demographic and clinical characteristics of participants

Parameter	HFN2-008 group	Placebo group	p-value1)
Number of cases	47	48
Sex (Male: Female)	20:27	14:34	0.174
Age (years)2)	32.87 ± 10.79	32.02 ± 10.80	0.701
Height (cm)2)	166.29 ± 8.64	165.43 ± 8.57	0.063
Weight (kg)2)	74.67 ± 8.83	74.21 ± 9.50	0.806
Body mass index (kg/m2)2)	26.92 ± 1.33	27.01 ± 1.28	0.734
Alcohol consumption (Yes/No)	34:13	32:16	0.802
Smoking (Yes/No)	9:38	10:38	0.682

⁽¹⁾ Chi-square test or Fisher’s exact test

⁽²⁾ Values are expressed as mean ± standard deviation

Baseline physical examinations, vital signs, and clinical laboratory test results revealed no clinically significant abnormalities in either group. Although significant differences were observed for a few parameters, all values were within normal reference ranges. These findings indicate that the two groups were generally homogeneous at baseline.

Efficacy outcomes

Primary efficacy outcomes

DEXA revealed that the HFN2-008 group after 12 weeks of intervention showed significantly greater decreases in trunk and total body fat mass compared with the placebo group (p < 0.05; Table 2; Fig. 2). Similarly, the percentages of trunk and total body fat were significantly decreased in the HFN2-008 group compared with the placebo group (p < 0.05). By contrast, no significant between-group differences were observed in fat mass parameters of the upper or lower extremities.

Table 2.

Changes in body fat mass and percentage from baseline to week 12, as assessed with dual-energy X-ray absorptiometry

Parameters		Body fat mass (g)			Percent body fat (%)
		HFN2-008 group (n = 42)	Placebo group (n = 44)	p-value1)	HFN2-008 group (n = 42)	Placebo group (n = 44)	p-value1)
Left Arm	At baseline	1,307.95 ± 282.89	1,386.98 ± 281.18	0.198	35.88 ± 9.87	37.05 ± 8.20	0.589
	At 12 weeks	1,301.81 ± 323.97	1,347.11 ± 262.83	0.477	35.70 ± 10.33	36.90 ± 8.15	0.766
	Change from baseline	−6.14 ± 169.16	−39.86 ± 196.99	0.492	−0.18 ± 2.37	−0.15 ± 2.61	0.961
	p-value2)	0.892	0.200	-	0.701	0.718	-
Right Arm	At baseline	1,360.21 ± 294.77	1,448.18 ± 231.85	0.127	35.25 ± 9.91	36.83 ± 7.70	0.560
	At 12 weeks	1,346.43 ± 324.25	1,448.16 ± 261.64	0.112	35.31 ± 10.31	36.78 ± 7.77	0.759
	Change from baseline	−13.79 ± 130.90	−0.02 ± 125.27	0.625	0.06 ± 2.25	−0.06 ± 1.77	0.794
	p-value2)	0.525	0.933	-	0.781	0.730	-
Left Leg	At baseline	3,886.69 ± 968.19	3,944.98 ± 832.16	0.710	33.40 ± 8.13	33.65 ± 6.77	0.872
	At 12 weeks	3,781.88 ± 949.23	3,901.70 ± 814.93	0.473	33.07 ± 8.14	33.61 ± 7.03	0.625
	Change from baseline	−104.81 ± 313.24	−43.27 ± 312.12	0.364	−0.32 ± 1.86	−0.04 ± 1.61	0.452
	p-value2)	0.040	0.342	-	0.326	0.852	-
Right Leg	At baseline	3,913.33 ± 1,016.27	3,960.02 ± 904.16	0.710	33.35 ± 8.34	33.54 ± 7.03	0.913
	At 12 weeks	3,817.57 ± 948.84	3,967.93 ± 812.37	0.431	32.99 ± 8.19	33.74 ± 6.94	0.688
	Change from baseline	−95.76 ± 308.16	7.91 ± 349.47	0.149	−0.36 ± 1.83	0.20 ± 1.68	0.138
	p-value2)	0.061	0.936	-	0.234	0.449	-
Trunk	At baseline	13,861.74 ± 2,449.52	13,634.95 ± 1,601.83	0.611	39.58 ± 6.23	39.17 ± 4.85	0.735
	At 12 weeks	13,436.62 ± 2,835.12	13,955.07 ± 1902.42	0.320	38.89 ± 7.02	39.90 ± 5.16	0.450
	Change from baseline	−425.12 ± 1,211.93	320.11 ± 1,127.92	0.004	−0.69 ± 2.55	0.73 ± 2.17	0.007
	p-value2)	0.042	0.075	-	0.090	0.031	-
Total	At baseline	25,320.33 ± 4,381.49	25,345.00 ± 2,954.33	0.976	36.03 ± 6.77	36.07 ± 5.24	0.979
	At 12 weeks	24,663.38 ± 4,868.69	25,587.41 ± 3,304.99	0.304	35.55 ± 7.17	36.44 ± 5.49	0.517
	Change from baseline	−656.95 ± 1,685.86	242.41 ± 1,496.47	0.010	−0.47 ± 1.76	0.37 ± 1.53	0.019
	p-value2)	0.024	0.320	-	0.104	0.118	-

Values are expressed as mean ± standard deviation

⁽¹⁾ Independent t-test or Wilcoxon Rank Sum Test. ⁽²⁾ Paired t-test

Fig. 2.

Changes in trunk and total body fat. A Body fat mass and (B) body fat percentage from baseline to week 12 were assessed with dual-energy X-ray absorptiometry. Data are presented as mean ± standard deviation (SD). **p < 0.01 and *p < 0.05 indicate significant differences compared with the placebo group

CT revealed that decreases in visceral and subcutaneous fat areas after 12 weeks were significantly greater in the HFN2-008 group compared with the placebo group (p < 0.05; Table 3, Fig. 3). Furthermore, within-group analyses revealed that total abdominal, visceral, and subcutaneous fat areas were all significantly decreased compared with baseline in the HFN2-008 group (p < 0.05). However, the visceral-to-subcutaneous fat area ratio did not show a statistically significant difference between the groups (p = 0.646).

Table 3.

Changes in total abdominal, visceral, and subcutaneous fat areas from baseline to week 12, as assessed with computed tomography

Parameters		HFN2-008 group (n = 42)	Placebo group (n = 44)	p-value1)
Total Abdominal Fat Area (cm²)	At baseline	480.70 ± 102.26	461.88 ± 83.13	0.351
	At 12 weeks	449.79 ± 121.51	463.31 ± 84.67	0.549
	Change from baseline	−30.91 ± 55.40	1.43 ± 48.69	0.080
	p-value2)	0.001	0.846	-
Visceral Fat Area (cm²)	At baseline	117.73 ± 42.44	110.73 ± 43.87	0.454
	At 12 weeks	107.82 ± 44.43	109.72 ± 43.21	0.685
	Change from baseline	−9.91 ± 20.06	−1.1 ± 17.37	0.030
	p-value2)	0.003	0.701	-
Subcutaneous Fat Area (cm²)	At baseline	362.96 ± 74.04	351.15 ± 55.57	0.404
	At 12 weeks	341.97 ± 86.49	353.59 ± 57.35	0.467
	Change from baseline	−20.96 ± 38.80	2.44 ± 34.56	0.007
	p-value2)	0.001	0.641	-
Visceral-to-Subcutaneous Fat Area Ratio	At baseline	0.30 ± 0.14	0.32 ± 0.13	0.473
	At 12 weeks	0.31 ± 0.09	0.31 ± 0.11	0.726
	Change from baseline	0.02 ± 0.13	−0.01 ± 0.08	0.646
	p-value2)	0.370	0.608	-

Values are expressed as mean ± standard deviation

⁽¹⁾ Independent t-test or Wilcoxon Rank Sum Test. ⁽²⁾ Paired t-test

Fig. 3.

Changes in total abdominal, visceral, and subcutaneous fat areas from baseline to week 12, assessed with computed tomography. Data are presented as mean ± standard deviation (SD). **p < 0.01 and *p < 0.05 indicate significant differences compared with the placebo group

Secondary efficacy outcomes

DEXA revealed that total lean body mass showed no significant differences between the HFN2-008 and placebo groups after 12 weeks of intervention (p = 0.449). Specifically, the total lean body mass changed from 44.53 ± 7.22 kg at baseline to 44.47 ± 7.36 kg at week 12 (mean change: -0.05 ± 1.15 kg) in the HFN2-008 group, and from 43.83 ± 6.94 kg at baseline to 43.61 ± 6.81 kg at week 12 (mean change: -0.22 ± 1.14 kg) in the placebo group. This demonstrates that the adipose tissue reduction achieved by HFN2-008 was not accompanied by any muscle or lean tissue loss. Furthermore, changes in waist circumference were significantly greater in the HFN2-008 group compared with the placebo group (p < 0.05; Table 4; Fig. 4). Body weight, BMI, and hip circumference decreased from baseline in both groups; however, no significant differences were observed between groups.

Table 4.

Anthropometric measurements from baseline to week 12

Parameters		HFN2-008 group (n = 42)	Placebo group (n = 44)	p-value1)
Body Weight (kg)	At baseline	73.96 ± 8.51	74.20 ± 9.91	0.809
	At 12 weeks	72.54 ± 8.92	73.52 ± 9.60	0.628
	Change from baseline	−1.42 ± 2.15	−0.68 ± 1.88	0.079
	p-value2)	< 0.001	0.014	-
Body Mass Index (kg/m²)	At baseline	26.85 ± 1.45	26.93 ± 1.35	0.730
	At 12 weeks	26.34 ± 1.75	26.69 ± 1.47	0.311
	Change from baseline	−0.51 ± 0.80	−0.24 ± 0.66	0.088
	p-value2)	< 0.001	0.016	-
Waist Circumference (cm)	At baseline	83.11 ± 6.51	82.43 ± 6.83	0.471
	At 12 weeks	81.57 ± 6.95	82.00 ± 6.76	0.948
	Change from baseline	−1.54 ± 3.27	−0.43 ± 2.39	0.042
	p-value2)	0.004	0.182	-
Hip Circumference (cm)	At baseline	102.04 ± 4.60	102.14 ± 4.97	0.962
	At 12 weeks	100.71 ± 4.54	101.20 ± 4.20	0.608
	Change from baseline	−1.33 ± 2.29	−0.94 ± 2.38	0.316
	p-value2)	0.001	0.006	-
WHR (Waist-to-Hip Ratio)	At baseline	0.82 ± 0.06	0.81 ± 0.07	0.599
	At 12 weeks	0.81 ± 0.06	0.81 ± 0.06	0.974
	Change from baseline	−0.01 ± 0.03	0.00 ± 0.03	0.269
	p-value2)	0.350	0.411	-

Values are expressed as mean ± standard deviation

⁽¹⁾ Independent t-test or Wilcoxon Rank Sum Test. ⁽²⁾ Paired t-test

Fig. 4.

Changes in waist circumference from baseline to week 12. Data are presented as mean ± standard deviation (SD). *p < 0.05 indicates a significant difference compared with the placebo group

Changes in lipid-related parameters (total cholesterol, HDL cholesterol, LDL cholesterol, and triglycerides), inflammatory markers (high-sensitivity C-reactive protein), metabolic hormones (leptin, insulin, and adiponectin), and energy metabolism-related factors (UCP1 and PGC-1α) did not differ significantly between the HFN2-008 and placebo groups after 12 weeks of intervention (p > 0.05). However, within-group analyses showed significant changes in leptin and insulin levels in the HFN2-008 group after 12 weeks compared with baseline (p < 0.05). In the placebo group, a significant change in insulin levels was also observed (p < 0.05). Additionally, both groups exhibited significant within-group changes in UCP1 and PGC-1α levels after 12 weeks compared with baseline (p < 0.05). However, these changes were not significant between groups (Table 5).

Table 5.

Metabolic regulatory hormones and energy metabolism-related factors from baseline to week 12

Parameters			HFN2-008 group (n = 42)	Placebo group (n = 44)	p-value1)
Metabolic hormones
Leptin (ng/mL)		At baseline	12.14 ± 9.21	11.81 ± 6.99	0.733
		At 12 weeks	10.87 ± 8.41	10.73 ± 7.62	0.816
		Change from baseline	−1.27 ± 4.03	−1.08 ± 5.94	0.769
		p-value2)	0.048	0.236	-
Insulin (uU/mL)		At baseline	9.02 ± 3.66	10.71 ± 7.55	0.746
		At 12 weeks	7.65 ± 5.23	7.55 ± 4.92	0.948
		Change from baseline	−1.38 ± 3.84	−3.16 ± 5.59	0.288
		p-value2)	0.025	0.001	-
Adiponectin (ng/mL)		At baseline	9,724.26 ± 5,098.63	11,544.64 ± 6,526.71	0.242
		At 12 weeks	9,246.88 ± 4,794.25	11,246.51 ± 6,559.40	0.273
		Change from baseline	−477.38 ± 1,778.52	−298.13 ± 2,728.21	0.718
		p-value2)	0.089	0.472	-
Energy metabolism-related factors
UCP1 (ng/mL)		At baseline	0.72 ± 0.72	1.01 ± 0.98	0.057
		At 12 weeks	1.76 ± 0.98	2.34 ± 1.72	0.147
		Change from baseline	1.05 ± 0.95	1.33 ± 1.67	0.498
		p-value2)	< 0.001	< 0.001	-
PGC-1a (ng/mL)		At baseline	0.59 ± 0.47	0.84 ± 0.64	0.061
		At 12 weeks	1.25 ± 0.72	1.65 ± 1.16	0.152
		Change from baseline	0.66 ± 0.58	0.81 ± 1.01	0.743
		p-value2)	< 0.001	< 0.001	-

Values are expressed as mean ± standard deviation

UCP1 uncoupling protein 1, PGC peroxisome proliferator-activated receptor gamma coactivator-1 alpha

⁽¹⁾ Independent t-test or Wilcoxon Rank Sum Test. ⁽²⁾ Paired t-test

Safety outcomes

Safety was assessed in 95 participants who consumed HFN2-008 at least once. No serious adverse events were reported during the study period. A total of four nonserious adverse events occurred in four participants in the HFN2-008 group; however, all events were assessed by clinical investigators as unrelated to the study product. The incidence of adverse events did not significantly differ between groups (p > 0.05).

Clinical laboratory findings revealed significant between-group differences in changes from baseline to week 12 for alkaline phosphatase, creatinine, and estimated glomerular filtration rate (p < 0.05). However, all values remained within normal reference ranges or were considered clinically insignificant. No significant between-group or within-group differences were observed for the other laboratory parameters. Urinalysis results, including specific gravity and pH, showed no significant changes between or within groups, and all values were within normal ranges. Pregnancy tests for human chorionic gonadotropin (hCG) conducted in women of childbearing potential were negative for all participants.

Analysis of vital signs demonstrated no significant between-group differences in systolic blood pressure, diastolic blood pressure, pulse rate, or body temperature after 12 weeks. Although some within-group significant changes were observed, these remained within normal physiological ranges and were not considered clinically meaningful. Furthermore, electrocardiographic and physical examinations revealed no abnormal findings in either group.

Overall, these results indicate that no clinically significant safety concerns related to the consumption of the study product were observed during the intervention period.

Discussion

The present randomized, double-blind, placebo-controlled study evaluated the clinical safety and effects of 12-week HFN2-008 supplementation on body fat distribution and abdominal fat accumulation. Participants in the HFN2-008 group exhibited significant decreases in body fat mass and percentage compared with the placebo group, particularly in trunk and total body fat parameters. Furthermore, CT-based analyses revealed significant decreases in visceral and subcutaneous fat areas. These findings suggest that HFN2-008 may exert beneficial effects on body fat accumulation, especially in metabolically relevant abdominal fat depots.

Excessive accumulation of body fat, particularly abdominal and visceral fat, has been closely associated with insulin resistance, dyslipidemia, chronic low-grade inflammation, and increased risk of cardiovascular disease. Previous studies have emphasized that decreases in body fat mass (especially visceral fat) are more strongly associated with improved metabolic health compared with overall weight loss alone [15]. In this context, the CT-based decreases in visceral and subcutaneous abdominal fat observed in the present study represent clinically meaningful outcomes. Although absolute reductions were observed in both fat compartments, the visceral-to-subcutaneous fat area ratio did not change significantly (p = 0.646). This indicates that HFN2-008 promotes a concurrent and proportional reduction in both major abdominal fat depots, rather than selectively depleting one over the other.

DEXA showed that the HFN2-008 group underwent significantly greater decreases in trunk and total body fat mass and percentage after 12 weeks compared with the placebo group. Conversely, no significant between-group differences were observed in extremity fat mass. This pattern suggests that HFN2-008 does not exert fat-decreasing effects uniformly throughout the body but may preferentially target central fat depots with more pronounced fat accumulation. This observation is consistent with trends reported in previous human intervention studies investigating functional foods or bioactive compounds targeting adiposity [16, 17].

The improvements in abdominal fat indices confirmed by CT represent a key clinical implication of this study. Visceral fat is recognized as a metabolically active tissue that contributes to metabolic dysfunction through increased secretion of inflammatory cytokines and free fatty acids [18]. Decreases in visceral fat have been reported to improve metabolic health independently of changes in body weight [19]. Notably, the present study demonstrated improvements in abdominal fat parameters without significant changes in body weight or BMI. The specific mechanisms underlying these effects are not yet fully understood, but prior studies indicate that heat-treated B. longum and its metabolites can alter the gut environment to increase short-chain fatty acid (SCFA) production. Increased SCFAs subsequently activate AMP-activated protein kinase (AMPK) in peripheral tissues to promote fatty acid oxidation and inhibit lipid synthesis. In addition, these postbiotic preparations help strengthen the intestinal barrier. This enhanced barrier function decreases the systemic influx of lipopolysaccharides (LPS), which in turn alleviates the low-grade inflammation closely linked to visceral fat accumulation and metabolic dysfunction.

Lean body mass did not significantly differ between groups, indicating that the loss of body fat was not accompanied by a loss of muscle mass. Preservation of lean mass alongside decreases in fat mass is considered a favorable change in body composition and is an important criterion for evaluating the clinical utility of functional foods intended for body fat management [20, 21].

Waist circumference is a simple yet reliable clinical indicator of visceral fat accumulation [22], and a significantly greater decrease in waist circumference was observed in the HFN2-008 group compared with the placebo group. This finding is consistent with the imaging-based findings, further supporting the notion that HFN2-008 specifically decreases abdominal fat.

By contrast, changes in lipid-related parameters (total cholesterol, LDL cholesterol, HDL cholesterol, and triglycerides), inflammatory markers (high-sensitivity C-reactive protein), metabolic hormones (leptin, insulin, and adiponectin), and energy metabolism-related factors (UCP1 and PGC-1α) did not significantly differ between groups. This may be attributed to the relatively healthy study population, in whom most metabolic parameters were within or near normal ranges at baseline. Indeed, previous studies have reported that improvements in metabolic biomarkers are more pronounced in individuals with metabolic abnormalities or high-risk profiles [23, 24]. Moreover, the present study did not directly assess changes in gut microbiota composition or mechanistic biomarkers related to energy metabolism. Therefore, mechanistic interpretations of the observed fat-decreasing effects should be cautious and remain limited to plausible explanations based on previously reported physiological activities of postbiotics and heat-treated probiotic preparations.

From a safety perspective, no serious adverse events were reported during the study period, and all nonserious adverse events were considered unrelated to HFN2-008. Clinical laboratory tests, urinalysis, vital signs, electrocardiography, and physical examinations revealed no clinically meaningful abnormalities, indicating that 12-week HFN2-008 supplementation is generally safe and well tolerated [25, 26].

This study has several limitations. First, the study protocol was registered retrospectively due to an administrative oversight. We registered the study before the data lock and unblinding to prevent any post-hoc modifications and minimize the risk of outcome-driven reporting. However, retrospective registration still limits the transparency expected of clinical research and introduces a potential risk of reporting bias. Furthermore, the study evaluated multiple adiposity-related primary outcomes without prespecified multiplicity adjustments, which may increase the risk of Type I errors. Therefore, all efficacy findings should be interpreted as exploratory and hypothesis-generating rather than confirmatory. Second, we did not quantitatively measure habitual dietary intake or physical activity levels during the intervention. Participants were simply instructed to maintain their usual lifestyles. Because body composition is highly sensitive to diet and exercise, we cannot entirely rule out the possibility that undetected lifestyle changes influenced the results. Finally, the 12-week intervention period is relatively short, and we did not perform direct mechanistic evaluations such as gut microbiota profiling or SCFA quantification. Future long-term studies with rigorous dietary and physical activity tracking, as well as microbiome analyses, are needed to confirm our findings.

Conclusions

This human intervention study demonstrated that 12-week HFN2-008 supplementation significantly decreased body fat mass, body fat percentage, and visceral abdominal fat area, independently of changes in body weight. Notably, the selective decrease in adipose tissue without a concomitant loss of lean body mass indicates a qualitative improvement in body composition, and the absence of adverse effects confirms its favorable safety profile. These findings demonstrate that HFN2-008 has potential as a functional ingredient for body and abdominal fat management. Further studies involving populations with metabolic abnormalities and longer intervention periods are warranted to elucidate the underlying mechanisms of action.

Abbreviations

IP

Investigational product

ITT

Intention-to-treat

PP

Per-protocol

Authors' contributions

Conceptualization, M.B. and H.G.L.; methodology, H.C. and Y.G.K.; validation, H.C. and M.R.J.; formal analysis, H.C.; investigation, M.B.; resources, M.B.; data curation, S.K.O. and C.R.P.; writing—original draft preparation, H.C.; writing—review and editing, H.G.L.; visualization, S.K.O.; supervision, H.G.L. and M.B.; project administration, M.R.J.; funding acquisition, Y.G.K. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by Huons-N Co., Ltd., grant number HOS-BF-2024, which also provided the investigational product.

Data availability

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Declarations

Consent for publication

Not applicable.

Competing interests

Y.G.K., M.R.J., S.K.O. and C.R.P. are employees of Huons N Co., Ltd., the sponsor of this study and provider of the investigational product. While these sponsor-affiliated authors contributed to funding acquisition, project administration, and basic data curation, they had no role in the clinical study execution, primary statistical analysis, clinical interpretation of the outcomes, or the final decision to publish the results. The independent academic and clinical investigators retained complete control over the study data, its objective interpretation, and the drafting of the manuscript. The remaining authors declare no competing interests.