**3.** *Hafnia alvei* **HA4597® reduces body weight gain, fat mass and food intake in murine models of obesity**

The first preclinical study was conducted to validate *H. alvei* and its production of ClpB, as a potential probiotic and its postbiotic for appetite and body weight management in overweight and obesity. Legrand *et al.* [14] tested *H. alvei* HA4597® on two

mouse models of obesity: genetic, leptin deficient ob/ob mice (a model of hyperphagia) and nutritional, High-Fat Diet (HFD)-induced obesity.

This study confirmed that *H. alvei* HA4597® significantly reduces body weight gain and fat mass in both models of obese mice (**Figure 3a**-**d**). In the hyperphagic ob/ob model, *H. alvei* HA4597® treatment significantly reduces food intake with a 20.8% decrease (vs placebo) in total intake measured after 18 days (p < 0.001), the difference becoming significant from day 8 (**Figure 3e**) and was accompanied by a higher level of phosphorylated hormone-sensitive lipase in fat tissues (p < 0.01) (**Figure 3g**).

The effect of *H. alvei* on food intake was not observed in mice on HFD, considering that HFD models tend to hypophagia because mice are not attracted to fat (**Figure 3f**).

Thus, *H. alvei* HA4597® exhibits the desired probiotic properties of an appetite and body weight management supplement i.e., it triggers anorexigenic and lipolytic effects in hyperphagic mice resulting in decreased body weight gain and fat mass.

A second trial conducted by Lucas *et al.* [17] evaluated the efficacy of *Hafnia* on another animal model of obesity based on a combined model of both HFD-fed and genetic ob/ob mice that may represent most closely hyperphagia and diet-induced obesity in humans (compulsive eating behavior combined with hypercaloric diet and accompanied by functional leptin resistance). This study also compares the efficacy of the strain to the drug Orlistat, a lipase inhibitor used in humans for the management of obesity.

A daily provision of 1.4 x 1010 Colony Forming Units (CFU) of *H. alvei* HA4597® in these mice significantly decreased the food intake, the body weight gain (**Figure 4A** and **B**) and total fat mass and preserved lean mass, resulting in an improved lean/fat mass ratio (**Figure 4C**). On top of that, other metabolic parameters were improved with the *H. alvei* treatment, including glycemia, total cholesterol and hepatic alanine aminotransferase (ALAT) (**Figure 4E**).

Although Orlistat is effective for weight loss, in contrast to *H. alvei* HA4597®, it is accompanied in this study by a strong hyperphagic effect which may be the cause of the increased glycemia observed for this group (**Figure 4D**).

After *H. alvei* proved successful in the regulation of appetite and weight in mice models, the next step was to evaluate efficacy in humans.

*H. alvei* HA4597® improves weight loss in a multicentric, double-blind, randomized placebo-controlled trial including 236 overweight adults.

To **investigate the clinical efficacy** of the strain, a 12-week prospective, doubleblind, randomized study was realized on 236 overweight men and women (230 followed protocol and were analyzed in the per protocol results) [18]. All subjects were on a − 20% low-caloric diet and were asked to maintain their usual physical activity. Subjects received either 2 capsules per day providing 1011 bacteria of *H. alvei* HA4597® per day (HA) or a placebo (P).

The primary outcome was the percentage of subjects losing 3% or more of their body weight after 12 weeks. Indeed, **significantly more subjects (+38%) lost at least 3% of their initial weight** after 12 weeks than in the placebo group (57.7 vs. 41.7%, p = 0.028, Per Protocol results) (**Figure 5A**). 51% more participants also lost more than 4% of their body weight on HA than on placebo (46.2% vs. 30.6%, p = 0.024) (**Figure 5B**). The average weight loss observed in the treated group was 3.6% at 12 weeks, vs. 2.9% in the placebo group, a high figure linked to the strong effect of the hypocaloric diet.

*The Potential of Precision Probiotic* Hafnia alvei *HA4597 to Support Weight Loss DOI: http://dx.doi.org/10.5772/intechopen.103723*

### **Figure 3.**

*Results of* Hafnia alvei *HA4597® supplementation vs. control in Ob/Ob mice and HFD mice models of obesity (adapted from Legrand et al., 2020). Body weight dynamics in ob/ob (a) and in HFD-fed obese mice (b), Two-way RM ANOVA, p < 0.001, (b) \*for days 23–25, 38, 41, and 47, \*\*for days 26, 27, 30, 37, 39, 42, and 43, \*\*\*for days 28, 29, 31–36, and 40. Total fat and lean tissue mass in ob/ob (c) and in HFD-fed obese mice (d). Mann–Whitney tests, \*\*p < 0.01, \*p < 0.05. Cumulative food intake in ob/ob (e) and in HFD-fed obese mice (f), Two-way RM ANOVA, p < 0.05, Bonferroni post tests, \*\*\*p < 0.001, \*\*p < 0.01, \*p < 0.05. Actin-normalized pHSL levels in the epididymal fat tissue in ob/ob (g), Mann-Whitney tests, \*p<0.05.*

### **Figure 4.**

*Results on body weight gain and other parameters after 37 days of oral gavage with* Hafnia alvei *HA4597® vs. orlistat in HFD Ob/Ob mice (adapted from Lucas et al., 2020). (A) Cumulative food intake (g). Two*≃*way RM ANOVA, p < 0.0001, Bonferroni's posttests, HFD vs. HFD + Orlistat. \*\*\* p < 0.001; \*\* p < 0.01 days 24,25; \* p < 0.05 days 22,23. HFD + H. alvei vs. HFD + Orlistat. \*\*\* p < 0.001, \*\* p < 0.01, days 17,18; \*p < 0.05 days 15,16. (B) Body weight gain (%). Two*≃*way RM ANOVA, p < 0.0001, Bonferroni's posttests, HFD vs. HFD + Orlistat, \*\*\* p < 0.001,\*\* p < 0.01, \* p < 0.05; C. ANOVA, p < 0.0001, Tukey's posttests avs. HFD and bvs. HFD + H. alvei, both \*\*\* p < 0.001, Student's t*≃*tests, \* p < 0.05, (mean ± SEM; SDiet, n = 16, all other groups, n = 24). (C) Lean/fat mass ratio at the end of the treatment. Student's t*≃*tests, # p < 0.05, (mean ± SEM; SDiet, n = 16, all other groups, n = 24). (D) Basal glycemia at end of treatment (g/L). ANOVA p < 0.05, Tukey's posttest vs. SDiet \* p < 0.05. Student's t*≃*test # p < 0.05. (E) Alanine transaminase (ALAT) levels at end of treatment (U/L). ANOVA p = 0.0006, Tukey's posttests \*\* p < 0.01, \* p < 0.05, (mean ± SEM; SDiet, n = 8, all other groups n = 12).*

*The Potential of Precision Probiotic* Hafnia alvei *HA4597 to Support Weight Loss DOI: http://dx.doi.org/10.5772/intechopen.103723*

### **Figure 5.**

*Results of the clinical study with 12-week supplementation of overweight subjects with* Hafnia alvei *HA4597® or placebo (adapted from Déchelotte et al., 2021, except Figure 5D, unpublished). (A) Proportion of subjects who lost at least 3% of body weight after 12 weeks PP population, Exact Fisher's test P. vs HA. \*p≤0.05. (B) Proportion of subjects who lost at least 4% of body weight after 12 weeks PP population, Exact Fisher's test P. vs HA. \*p≤0.05. (C) Hip circumference change vs. T0 ITT population, Mann-Whitney-U test (w12-w0) P. vs. (w12-w0) HA. \*pU≤0.001. (D) Serum glucose concentration before and after supplementation ITT population, Mann-Whitney-U test (w12) P. vs. (w12) HA. \*pU ≤ 0.05. (E) Feeling of fullness ITT population, Mann-Whitney-U test (w12) P. vs. (w12) HA.\*\*pU ≤ 0.01. (F) Change in Feeling of fullness ITT population, Mann-Whitney-U test; (w12-w0)P. vs. (w12-w0)HA.\* pU ≤ 0.05. Paired Wilcoxon test; HA(w0) vs. HA(w12).\*\* pwi ≤ 0.01.*

Compared to the placebo group, the participants in the probiotic group saw a significant further 1.2 cm reduction in **hip circumference** (p < 0.001 at 12 weeks) as well as a significant decrease in **blood glucose** (p = 0.027 at 12 weeks) (**Figure 5C** and **D**). Furthermore, the HA group recorded a significant decrease of **cholesterol** compared to the beginning of the study (p = 0.008 for total cholesterol and p = 0.028 for LDLcholesterol at 12 weeks) (unpublished).

Importantly, this study also revealed an **increased feeling of fullness** assessed by visual analog scale (VAS) (p = 0.009 at 8 weeks) in the HA group (**Figure 5F**). In the VAS, a score of 50 means that there is no feeling of hunger. Both groups were under a − 20% hypocaloric diet, but only the treated group led to feeling of fullness scores above 50 (**Figure 5E**). This confirms the mechanism of action of this precision probiotic through the regulation of appetite and this led, despite the constraints of the diet, to a significantly higher level of satisfaction of the subjects from the treated group, compared to the placebo group.

Indeed, in the HA group, benefit of treatment was rated as "very good" or "good" by 67.9% of subjects compared to 53.1% in the placebo group (p = 0.019). Only 5% of subjects in the probiotic group rated it as "poor", as opposed to 14.2% in the placebo group.

This level of satisfaction shows the users have a clear perception of efficacy, and the good appetite regulation during a diet makes it easier to keep compliant to the reduced caloric intake, without the difficulty and discomfort associated with hunger.

According to the official guidelines for the management of obesity, a 3 to 5% weight loss is the reference interval associated with meaningful clinical benefits: reductions in serum triglycerides concentrations, of blood glucose, HbA1C, and the risks of developing type 2 diabetes [19].

With clinically relevant efficacy as soon as in the first 12 weeks, excellent tolerability and no adverse events, **these results support the use of** *H. alvei* **HA4597® in the global management of excess weight.**
