Supplementation of Heat-Treated Lactiplantibacillus plantarum nF1 Changes the Production of Short-Chain Fatty Acids in Healthy Infants

Background Imbalance of the gut microbiome and decrease in the number of short-chain fatty acid (SCFA)-producing bacteria often affect human health by altering intestinal and immune homeostasis. The use of probiotics has been shown to be an attractive method to modulate gut microbiota to prevent or treat intestinal dysbiosis. Likewise, this study aimed to determine whether the oral consumption of heat-treated Lactiplantibacillus plantarum nF1 (HLp-nF1) induces changes in the gut environment in healthy infants by measuring changes in fecal SCFAs. Methods The study enrolled 43 infants aged under 2 months, with 30 infants in the HLp-nF1 group receiving HLp-nF1 orally (2.5 × 1010 cells/g/pack, daily dose of two packs) for 8 weeks. The fecal samples were collected and the questionnaires were administered at weeks 0 and 8. Results The concentrations of the total SCFAs, acetate, propionate, and butyrate significantly increased following HLp-nF1 supplementation (P < 0.0001, P < 0.0001, P < 0.0001, and P=0.028, respectively). Conclusions Supplementation of HLp-nF1 has a positive effect on SCFA production and could be a potentially useful and straightforward method to manipulate SCFA formation.


Introduction
Te human gastrointestinal tract comprises a complex and dynamic microbial community, and reportedly, the gut microbiota is critical for numerous aspects of human health [1].It modulates innate immunity, protects against pathogens by maintaining the intestinal mucosal barrier or providing anti-infammatory signals to the host, and regulates metabolic homeostasis via essential nutrient synthesis and absorption [1][2][3][4][5][6][7].
One of the main functions of the gut microbiota is the metabolic ability to transform complex polysaccharides into simple sugars, which are fermented to form short-chain fatty acids (SCFAs) [8,9], the main metabolites produced by the microbiota in the colon.Tey are defned as volatile saturated fatty acids with one to six carbon atoms in the aliphatic chain, existing in either a straight or branched conformation [9][10][11].Te major SCFAs produced are acetate, propionate, and butyrate [7,[12][13][14].Tese acids need to be produced in adequate amount to maintain the gut health and the well-being of the host [9,15].SCFAs play important roles both directly and indirectly in regulating immune and intestinal homeostasis, provide the primary source of energy for colonocytes [16,17], reinforce intestinal barrier function [18], regulate the immune system through various signaling pathways [9,[19][20][21][22], and control glucose or lipid metabolism [23][24][25].
Dysbiosis, a disturbance in the gut microbiota composition, results in a decrease in the number of bacteria producing SCFAs.In particular, infancy characterizes a highly dynamic stage wherein gut microbiota form and develop under the infuence of various factors [3].Te benefcial efect of probiotics on the balance of gut microbiota and the production of metabolites, including SCFAs, has been confrmed by the results of numerous studies [9].Recently, the interest and application of inactivated strains or dead cells, called postbiotics, have been increased due to their safety and storage capacity [26].A postbiotic is defned as a "preparation of inanimate microorganisms and/or their components that confers a health beneft on the host" [27][28][29][30].In the present study, we aimed to determine whether oral consumption of heat-treated Lactiplantibacillus plantarum nF1 (HLp-nF1) induces changes in the gut environment in healthy infants by measuring changes in fecal SCFAs.

Materials and Methods
2.1.Subjects.We recruited infants aged <2 months, born at the CHA Bundang Medical Center (Seongnam, Republic of Korea) and Kangwon National University Hospital (Chuncheon, Republic of Korea) between November 2021 and February 2022.Te inclusion criteria were: infants born (1) between 37 and 42 weeks of gestation; (2) with a birth weight between 2,500 and 4,500 g; and (3) without any prenatal and postpartum adverse events.We excluded infants: (1) diagnosed with congenital malformations or chromosome abnormalities; (2) with a history of gastrointestinal diseases; (3) admitted after birth; (4) with a history of using systemic steroids or antibiotics; (5) exposed to foods other than breastmilk or formula; and (6) whose parents did not consent to a stool test.
Tis study included healthy volunteers.Infants whose parents consented to the intake of HLp-nF1 for 8 weeks were classifed as the HLp-nF1 group, while the remaining infants were included in the control group.

Study Design.
Te duration of the study was 8 weeks.HLp-nF1 was manufactured by incubating L. plantarum nF1 for 20 h under pH control and then sterilized at 80 °C for 10 min [31].Infants in the HLp-nF1 group orally received a daily dose of two packs of HLp-nF1 (2.5 × 10 10 cells/g/pack) (one pack in the morning and one pack in the afternoon with feeding).Infants in the control group did not receive additional experimental treatment.During the study, all subjects were instructed not to consume other probiotics or change their formula.Fecal samples and questionnaires regarding weight, height, delivery information, feeding mode, stool characteristics, and gastrointestinal symptoms were collected at weeks 0 and 8. Tis study was approved by the Institutional Review Board (IRB) of the Ethics Committee of CHA Bundang Medical Center (IRB no.2021-07-077).Written informed consent was provided by the parents of infants.
2.3.SCFA Analysis.Fecal samples were collected from diapers using sterile swabs, immediately transferred to sterile cryogenic tubes, and stored in a −20 °C freezer until delivery to the laboratory.
SCFAs were extracted from fecal samples (50 mg) using deionized water (800 µL) and 5 M HCl (10 µL).After brief vortexing, 400 µL of ether was added to the samples, followed by mixing and shaking in the refrigerator for 5 min.After centrifugation (14,000 rpm, for 1 min), 20 µL of N, O-Bis (trimethylsilyl) trifuoroacetamide was added to 200 µL of ether layer, and the mixture was incubated at 70 °C for 20 min and at 37 °C for 2 h.Subsequently, the derivatives of SCFAs were assessed.
Gas chromatography analysis was performed using GC-2010 Plus, GCMS-TQ 8030 (Shimadzu, Tokyo, Japan) with a DB-5 ms column (inner diameter: 30 mm × 250 mm; flm thickness: 0.25 μm; Agilent J&W Scientifc, Folsom, CA, USA).Te gas chromatography conditions were as follows: 1 µL of derivatives was injected in split mode with a ratio of 50 : 1; injection temperature: 200 °C; and column oven temperature: 40 °C.Te initial temperature was 40 °C and maintained for 2 min; thereafter, it was increased from 40 °C to 70 °C at a rate of 10 °C/min, from 70 °C to 85 °C at a rate of 4 °C/min, from 85 °C to 110 °C at a rate of 6 °C/min, and fnally from 110 °C to 290 °C at a rate of 90 °C/min; this temperature was maintained for 6 min.Helium was used as a carrier gas at a constant fow rate of 0.89 mL/min through the column.Te temperature of the electron impact ion source and interface were set to 200 °C and 250 °C, respectively.Te detector energy was 0.1 kV, event time was 0.03 s, and the mass spectrum data were collected in scan mode (m/z 117: acetic acid, 131: propionic acid, 145: butyric acid).Te concentrations of SCFAs were calculated using a standard solution of butyric acid (B103500; Sigma-Aldrich Co., St. Louis, MO, USA), propionic acid (94425; Sigma-Aldrich Co.), and acetic acid (31010S0350; JUNSEI Chemical Co., Tokyo, Japan).

Statistical Analysis.
Data were analyzed using descriptive statistics and presented as means with standard deviations, or as medians with an interquartile range (IQR), and proportions.Comparisons of the mean values of continuous and categorical variables between groups were conducted using the t-test and Fisher's exact-test, respectively.Linear regression coefcient analyses were used to calculate unstandardized regression coefcients.P values <0.05 de-noted statistically signifcant diferences.All statistical analyses were performed using IBM SPSS ® Statistics 28.0.1.1 (IBM Corporation, Armonk, NY, USA).

Comparison of Baseline Characteristics between Control and HLp-nF1
Groups.Forty-three healthy volunteers were included in this study.Te number of subjects included in the control and HLp-nF1 groups was 13 and 30, respectively.Baseline characteristics, including sex, birth information, feeding mode, and age, did not vary signifcantly between the two groups (Table 1).

Comparison of Questionnaire Data after 8 Weeks between
Control and HLp-nF1 Groups.In this study, there were no cases of withdrawal due to side efects or loss at follow-up.Hence, a total of 43 subjects completed the questionnaire after 8 weeks.Tere was no signifcant diference between the two groups in any of the questionnaire items (Table 2).Mean weight and height after 8 weeks were 6,200 g and 62.04 cm in the control group, and 6,285.18g and 60.57cm in the HLp-nF1 group, respectively.Weight and height growth assessment was properly performed in both groups.More than half of the cases did not report gastrointestinal symptoms.Defecation was more frequent in the HLp-nF1 group versus the control group, but the diference was not statistically signifcant.

Concentration of SCFAs and Efect of HLp-nF1
Supplementation.We performed two measurements of SCFA metabolites at weeks 0 and 8 to observe changes that occurred during the supplementation of HLp-nF1.Acetate was the most abundant SCFA, followed by propionate and butyrate.Te concentrations of total SCFAs, acetate, propionate, and butyrate were signifcantly increased after HLp-nF1 supplementation (P < 0.0001, P < 0.0001, P < 0.0001, and P � 0.028, respectively).Although the same trend was observed in the control group, the results were not statistically signifcant (Table 3, Figure 1).
Linear regression coefcient analyses of diferences in the concentration of SCFAs in association with other variables in the HLp-nF1 group were performed to investigate the efect of HLp-nF1 supplementation.Other variables (i.e., sex, age, birth mode, birth weeks, birth weight, birth height, birth head circumference, and feeding mode) were not signifcantly associated with changes in SCFA concentration, except for birth weeks at the propionate analysis (Table 4).

Discussion
In this study, we compared changes in the gut environment of healthy infants after the administration of HLp-nF1 for 8 weeks by monitoring clinical response and fecal SCFAs.First, adequate growth was observed in both groups, and safety was demonstrated.Second, SCFA production was signifcantly increased in the HLp-nF1 group compared with the control group.
In terms of clinical response, there were no cases of withdrawal due to side efects or gastrointestinal symptoms reported during the study period.Growth, the most important factor in infants, was appropriate in terms of height and weight.Safety is one of the most important criteria in the selection of probiotic strains for human consumption.In particular, HLp-nF1 is nonviable heat-killed microorganism, which exhibits a low risk of sepsis or bacteremia associated with probiotics [32][33][34][35][36][37], particularly in critically ill or vulnerable patients and pediatric populations, demonstrating comparable efects to those of viable probiotics [26,36,[38][39][40].
Acetate, propionate, and butyrate account for 85%-95% of the total SCFAs in all regions of the colon [9,41].Notably, acetate is the most abundant SCFA, accounting for >50% of the total SCFAs [9,42].In accordance with previous studies, the concentration of acetate was the highest among all SCFAs in this study.However, the baseline values of fecal SCFAs and the diferent age-or diet-related patterns of change in SCFAs remain unknown [43].In one study measuring fecal SCFAs at birth, day 30, and day 60 in healthy term infants from diferent feeding groups (i.e., extensively hydrolyzed formula, amino acid formula, or human milk), there was no signifcant trend with increasing age up to day 60.In the human milk group, the average concentration of total SCFAs at birth, day 30, and day 60 was approximately 40 mmol/g, similar to that of acetate; the average concentration of propionate and butyrate was <2 μmol/g [44].Tese values were similar to the SCFA concentration measured in the control group at week 8 in our study.While both groups showed an increasing trend in SCFA concentrations after 8 weeks, the diference was not statistically signifcant in the control group.Furthermore, other factors that could afect the SCFA concentration were evaluated by performing linear regression coefcient analyses.Te results of these analyses confrmed the relationship between diferences in the concentration of SCFAs and other variables, including sex, age, birth mode, birth weeks, birth weight, birth height, birth head circumference, and feeding mode.
It has been shown that SCFAs play a crucial role in maintaining intestinal and immune homeostasis, particularly in regulating the maturation, integrity, and function of the gut barrier [9,43].After supplying colonocytes, SCFAs are transported from the intestinal cavity into the blood vessels and fnally to organs as substrates or signaling molecules to perform numerous physiological functions [25].In addition, since 95% of SCFAs are reabsorbed or metabolized by gut microbiota, small changes in SCFA concentration in fecal excretion actually represent large diferences in production in the gut [12].Terefore, the signifcant increase in SCFAs recorded after 8 weeks of HLp-nF1 intake can be considered meaningful.
While SCFA is infuenced by multiple factors, the most signifcant mechanism inducing a meaningful change in SCFA concentrations following HLp-nF1 consumption can be attributed to alterations in the composition of gut microbiota.In the microbiome analysis of 30 infants within the HLp-nF1 group, altered bacterial composition after HLp-nF1 intake was observed; marked increases were noted in benefcial bacteria, such as Bifdobacterium and Veillonella, while decreases were observed in certain opportunistic pathogens, including Prevotella, Streptococcus, and Sutterella (Figure S1, Table S1).Although a comprehensive analysis of   gut microbiota composition was not conducted in this study, we hypothesize that ingestion of HLp-nF1 may impact rebiosis, the re-establishment of the native microbiota, by serving as a barrier and impeding the colonization of opportunistic bacteria [45].Especially, Lactiplantibacillus strains enhance the integrity of the intestinal barrier, which may afect decreasing translocation of bacteria across the intestinal mucosa [46].One possible reason for the absence of an increase in the relative abundance of Lactiplantibacillus per se is thought to be the low quantity and short duration of HLp-nF1 administered relative to the total gut microbes.Nevertheless, this regulation of the gut environment induced by the supplementation of HLp-nF1 may have afected the increase in SCFAs by controlling the ratio of benefcial bacteria to opportunistic pathogens.Previous studies on L. casei or L. plantarum also showed similar alterations in the   [14,15,49,50].Veillonella produces propionate through the succinate pathway [51][52][53].Human milk oligosaccharides in breastmilk and galactooligosaccharides or fructo-oligosaccharides in infant formula possess prebiotic properties that allow these bacteria to efectively produce SCFAs [54,55].In addition to these mechanisms, exopolysaccharides (EPS) (the polysaccharides synthesized and secreted by bacteria) may also be a major source of SCFAs.Lactiplantibacillus and Bifdobacterium are the main EPS-producing strains, and they can degrade and ferment EPS into SCFAs [56,57].

Journal of Nutrition and Metabolism
A couple of study limitations should be noted.Firstly, the number of subjects in the control group was relatively small.Because the study was conducted with healthy infants who met various conditions, there was difculty in recruiting a sufcient number of subjects.Secondly, it was difcult to control all factors afecting the concentration of SCFAs.Te concentration and ratio of SCFAs are associated with the composition of the gut microbiome, diet, genetics, and other environment factors [9,48].However, subjects of a similar age who had a restrictive diet before initiating feeding with solid food were recruited.Factors that could exert an efect, such as birth mode, were similar between the two groups.
Nevertheless, the results of this study are signifcant because we confrmed that postbiotics intake for a short period of 8 weeks in infancy, when alterations in the gastrointestinal environment are very dynamic, induces changes in the production of SCFAs.Tis study is also meaningful in that it is the frst study of changes in SCFAs using HLp-nF1 to the best of our knowledge.Further research and long-term follow-up studies in various species and numerous strains are warranted to better understand the efects of postbiotics administration on the production of specifc SCFAs.

Conclusions
Tis study demonstrated that an 8-week oral administration of HLp-nF1 may increase SCFA production in healthy infants.It can be suggested that the use of postbiotics is a useful and easy method for manipulating SCFA formation by alterations in the human gut microbiota.Tis approach can be utilized to prevent or treat intestinal dysbiosis.

Figure 1 :
Figure 1: Box plots of the concentrations of SCFAs in the HLp-nF1 group.

Table 1 :
Baseline characteristics of children in the control and HLp-nF1 groups.Data are presented as numbers (percent) or means ± SD.Comparisons of mean values of continuous and categorical variables between groups were conducted using the t-test and Fisher's exact-test, respectively; P values <0.05 denote statistically signifcant diferences.C/S, cesarean section; HLp-nF1, heat-treated Lactiplantibacillus plantarum nF1; NSVD, normal spontaneous vaginal delivery; SD, standard deviation.

Table 2 :
Questionnaire after 8 weeks in the control and HLp-nF1 groups.
Data are presented as numbers (percent) or means ± SD.Comparisons of the mean values of continuous and categorical variables between groups were conducted using the t-test and Fisher's exact-test, respectively; P values <0.05 denote statistically signifcant diferences.HLp-nF1, heat-treated Lactiplantibacillus plantarum nF1; SD, standard deviation.

Table 3 :
Changes in SCFA concentration in the control and HLp-nF1 groups.

Table 4 :
Linear regression coefcients analyses of diferences in the concentration of SCFAs in association with other variables in the HLp-nF1 group.