Rectal administration of buttermilk processed with medicinal plants alters gut microbiome in obese individuals

Abstract

Objective

To evaluate the effect of rectal administration of buttermilk processed with medicinal plants on gut microbial composition and thereby on weight in obese individuals.

Methods

With ethics committee approval, 16 obese individuals in the age group 20–50 years (BMI ≥30 kg/m²) were recruited who received a course of 15-enemas over 15-days. Of these, 1st, 8th and 15th enemas were of sesame-oil administered after food, while other enemas were of buttermilk processed with medicinal plants administered before food. Outcome variables viz. anthropometry, body composition, blood glucose, insulin and lipid profile were evaluated on day 0, 16 and 45. Also, microbial composition of buttermilk preparation and faecal samples of patients collected on day 0, 16 and 45 were studied with the help of 16S rRNA gene sequencing.

Results

The circumferential measures and skinfold-thickness showed a decrease on day 16, which remained lower as compared to baseline till day 45. A gradual decrease in blood-glucose was seen, which was statistically significant on day 45, while insulin levels increased on day 16 and fell to baseline on day 45. There was an overall increase in bacterial diversity on day 16 that settled back to its original composition by day 45.

Conclusion

Our findings suggest that buttermilk administration per rectum is effective for a specific period and may have to be repeated for sustained benefits.

Supplementary Information

The online version contains supplementary material available at 10.1007/s40200-021-00879-z.

Introduction

Obesity and associated comorbidities have become epidemic worldwide. The energy imbalance in terms of increased intake of calories and decreased activity profile is the major contributing factor for the development of obesity [1]. Among various pathologies suggested, altered gut microbiota has been reported to be a key factor in the control of body weight along with energy metabolism [2]. The collective microbial community of the healthy human gut is very complex and consists of five dominant bacterial phyla viz. Firmicutes, Bacteroidetes, Actinobacteria, Proteobacteria, and Verrucomicrobia, along with less abundant phyla such as Cyanobacteria, Fusobacteria, and others [3].

The composition of gut microbiota in obese individuals differs from that in lean individuals. It has been observed that the individuals exhibiting adiposity, insulin resistance and dyslipidemia show decrease in bacterial richness [3]. Among the major phyla present, Firmicutes and Bacteroidetes represents 90% of gut microbiota. At genera level, Bifidobacterium and Lactobacillus, responsible for positive physiological effects like improvement in the barrier function of the intestinal mucosa, have shown a reduction in an animal model of obesity. Lactobacillus and Clostridium species are associated with insulin resistance. Of these, Lactobacillus has displayed a positive correlation with fasting glucose and HbA1c levels, while Clostridium has shown a negative correlation with these parameters [4]. It has been observed that gut microbiota modulated mucosal immune response and ‘protective gut flora’ could prevent or delay diabetes development [5]. Patil et al., 2012 analyzed and quantified the dominant gut microbiota of lean, healthy, obese and surgically treated obese individuals of Indian origin. Predominance of genus Bacteroides along with high archaeal diversity and faecal SCFA levels were found to be prominent among obese individuals; whereas, surgically treated obese individuals exhibited comparatively reduced Bacteroides and archaeal counts along with reduced faecal SCFAs [6]. Considering these reports, modulation of the human gut microbiome can certainly be considered as a potential therapeutic target for the management of obesity.

Different dairy products such as milk, yogurt etc. have shown a promising effect on body weight regulation due to their Probiotic effect [7]. Buttermilk is a traditional fermented dairy product having higher abundance of Firmicutes (96.2%) with only 3.8% contribution from other phyla like Proteobacteria (0.1%), Bacteroidetes (0.01%), Actinobacteria (0.01%) etc. In firmicutes, the represented genera were Lactobacillus, Streptococcus, Enterococcus, and Macrococcus [8].

Ayurveda, the Indian System of Medicine, is being explored through contemporary basic and applied sciences. A report by Chaudhari et al. revealed microbial diversity associated with different prakriti types [9]. Another study aimed towards profiling healthy gut microbiome structure within three predominant prakriti revealed signature microbes [10]. Furthermore, Eliot Steer explored the pathology of Major depressive disorder (MDD) and its association with gut dysregulation within both the Ayurvedic and Western systems of medicine [11]. Buttermilk is recommended in Ayurveda as a probiotic and a modulator of the human gut microbiome [12]. Although there are no references regarding per rectum administration of buttermilk for obesity management, buttermilk enemas are prescribed in Ayurveda for few gastrointestinal disorders [13].

With this background, in the present study, we administered buttermilk per rectum in obese individuals and evaluated its effect on various anthropometric and biochemical parameters along with gut microbiota composition. We processed the buttermilk with a mixture of 8 herbal drugs mentioned in Ayurveda and have used Ayurveda based protocol for the rectal administration.

Material and methods

The study was carried out between May 2018 and August 2018 with prior clearance from the Institutional Ethics Committee (BVDUCOA/EC/−3482/2017–18). It was registered in the Clinical Trial Registry of India (CTRI/2018/02/011859).

Inclusion and exclusion criteria

Obese individuals in the age group of 20 to 50 years of either sex with a BMI of ≥30 kg/m² were included in the study after obtaining their written informed consent. Patients with anal pathology which preclude per rectal drug administration viz. fissure, fistula etc., pregnant and lactating women and individuals with secondary obesity due to hormonal imbalance were excluded from the study.

Sample size

The present study was a pilot proof-of-concept study. The objective of the study was not to prove the efficacy, but rather to explore effect of enema on gut microbiota. Hence, a sample size of 16 was arbitrarily chosen without any statistical consideration. Considering 25% drop out rate, it was expected that minimum 12 individuals at the end of the study.

Study conduct

Once recruited, the participants were called to study site after 12 h of fasting. Each patient’s demographic details and medical history were recorded, followed by a physical and systemic examination. Subsequently, Anthropometric measures viz. height, weight, Waist Circumference (WC), Hip Circumference (HC), Neck Circumference (NC) and skinfold along with body composition analysis (body fat, visceral fat, subcutaneous fat & skeletal muscle mass) were measured.

Wall mounted Stadiometer was used to record the height. To measure weight, participants were asked to stand in an upright position with hands on sides on the digital scale without footwear. BMI was calculated according to the formula: BMI (kg/m²) = body weight (kg)/squared height (m²). WC was measured at the upper level of umbilicus HC was recorded around the widest portion of the buttock region. The point above thyroid cartilage was noted to measure NC. All circumferences were recorded using inextensible measuring tape. Waist to hip ratio (WHR) was calculated by dividing WC by HC. Skinfold thicknesses (SFT) were measured on the right side of the body at 4 sites viz. Biceps, Triceps, Subscapular & Supra-iliac using a Harpenden’s Skinfold Calliper following standard protocol [14].

Following this, 8 ml of blood was collected from the ante-cubital vein of each participant for estimation of biochemical parameters. The biochemical investigations viz. fasting glucose, fasting insulin and lipid profile were outsourced from an accredited laboratory. Leptin was estimated using ELISA kits (Ray Biotech, Inc., USA). The insulin resistance index was calculated by two methods viz. HOMA-IR [Sugar (mM/l) x Insulin]/405 and TG/HDL ratio. For microbiota analysis, faecal samples were collected in a sterile container and stored at −80 °C for further processing.

After completing the baseline assessment, all participants received a course of 15 enemas over 15 days (1 enema daily). Of these, 3 enemas (1st, 8th and 15th) were of sesame oil, which were administered after food in a quantity of 120 ml. The remaining enemas were of buttermilk processed with a mixture of 8 herbal drugs, namely Acorus calamus, Piper longum, Cyperus rotundus, Aegle marmelos, Anethum sowa, Randia spinosa, Trachyspe rmumammi and Saussurea lappa. In short, 320 ml of curd was churned and 80 ml of water was added to it. The plants, as mentioned above, were added to this buttermilk in a quantity of 10 g each. This processed buttermilk was administered to the patients on an empty stomach, i.e., before food. During the course of enemas, the participants were advised to follow a light diet as well as to avoid fried and spicy food. They were also asked to avoid late nights, day time sleep, suppression of natural urges [15] etc. After completion of all enemas, on day 16, all the assessments carried out at baseline were repeated.

The participants were then directly called after 30 days (day 45) for the final visit. No dietary or lifestyle modifications or medications for weight control were recommended during this period. Their blood sample was collected at this visit, and the clinical examination was repeated to assess whether the effect of buttermilk treatment is maintained after stopping the treatment. A stool sample was collected and stored at −80 °C until further processing for DNA extraction and sequencing.

Apart from the stool samples, the samples of plain buttermilk and buttermilk processed with herbal mixture were also subjected to microbiota analysis. The study participants were recruited in 3 batches to minimize the procedural errors. The batch-to-batch variation/consistency in microbial diversity in the buttermilk samples used for these 3 batches was also carried out.

DNA extraction and 16S rRNA gene sequencing

For the microbiome analysis, DNA was extracted from the collected stool sample and buttermilk using QIAamp® DNA Stool Kit (QIAGEN: Valencia, CA, USA). The extracted DNA was quantified and quality checked using Nanodrop and Agarose Gel Electrophoresis. V4 region of 16S rRNA gene was amplified using 515F (5′-GTGCCAGCMGCCGCGGTAA-3′) /806R (5′- GGACTACHVGGGTWTCTAAT-3′) universal primer set with sequencing adapters, as described in the Earth Microbiome Project (http://www.earthmicrobiome.org/). Further, the amplified products were purified and pooled to equimolar concentrations (PicoGreen) and sequenced using Illumina MiSeq platform as per the manufacturer’s instructions (Illumina technologies, USA) Sequencing was performed using V2 paired-end (2 × 250 bp) chemistry.

Sequence processing

A total of 1,324,840 raw 16S rDNA sequence reads were obtained for 65 faecal and buttermilk samples, with an average of 11,227 ± 9580 reads per sample. Adapter sequences were trimmed by using Cutadapt (1.18) tool and assembled using Paired-End reAdmergeR (PEAR) assembler [16, 17]. Quality trimming and removal of chimeric sequences and paired-end assembly was done using microbiome_helper. The 16S rRNA gene sequence data set was processed with Dada2 (v1.6.0) for filtering of noise, removal of singletons and chimera as well as for obtaining Amplicon Sequence Variant (ASV) with 100% sequence similarity [18]. During the above process, chimeric checking and abundance filtering were also monitored. ASVs with a number of the sequences <0.005% of the total number of sequences were discarded as recommended. The resulting ASV table was used for downstream analysis. The taxonomic assignment was performed based on SILVA database (version 132) in order to define which features best distinguish each taxonomic group [19]. Sequences which got assigned as members of Chloroplasts, Eukarya, Cyanobacteria, and Archaea were removed. The data was successfully submitted as a NCBI BioProject with Submission-ID: SUB7100510 and BioProject ID: PRJNA610497.

Diversity analysis and functional prediction

Microbial diversity shift over the three-time points (0th, 16th & 45th day) was monitored at phylum as well as genus level. Also, diversity change in buttermilk preparation was tracked by comparing the microbiota of buttermilk before and after the preparation. Further, functional predictions were made based on 16S amplicon data with the help of prediction models using the Piphillin Server [20] applicable to the output obtained from the SILVA dataset (version 132). Functionally important pathways like Glycolysis/Gluconeogenesis, Butanoate Metabolism, Fatty acid biosynthesis, Insulin Signalling, Carbohydrate metabolism etc. were studied.

Statistical analysis

In the case of anthropometry and biochemical parameters, the parametric data is presented as Mean ± standard deviation and analyzed using One-way ANOVA test while the non-parametric data are presented as median (range) and analyzed using Kruskal Wallis test. In case of the gut microbiota analysis, alpha diversity was calculated with Microbiome Analyst software [16] based on the sequence similarity of 100%, for observed ASVs, Chao1 estimator, Shannon diversity indices. Beta diversity was measured by Jaccard, Bray-Curtis, unweighted and weighted UniFrac distances. PERMANOVA (Permutated Analysis of Variance) was utilized to evaluate the differences among day 0, day 16, and day 45. These distances were visualized via Principal Coordinate Analysis (PCoA).

Results

A total of 16 participants were recruited in the study. Of the 15 patients who completed the study, 8 were females and 7 were males. The mean age (± SD) of the participants was 33 (± 8.4) years.

Physiological parameters

It was observed that the difference in weight, BMI, and body composition was negligible throughout the study. The circumferential measures and skinfold thickness showed some reduction after the therapeutic course, i.e., on day 16, which though showed increase on day 45, remained lower than baseline. None of these changes were statistically significant (Table 1).

Table 1.

Effect of buttermilk administration on anthropometry, body composition, and vitals

Parameters	Day 0	Day 16	Day 45
Anthropometry
Weight (Kg)	85.4 ± 12.2	84.84 ± 12.2	84.72 ± 12.2
BMI (kg/m2)	33.6 ± 3.7	33.39 ± 3.9	33.38 ± 3.7
Abdomen circumference (cm)	101(60–118)	99(58–116)	100.5 (57–117)
Waist circumference (cm)	107 ± 11.1	101.9 ± 11.0	102.2 ± 11.61
Hip circumference (cm)	116 ± 9.1	111 ± 10.7	112.4 ± 10.1
Neck circumference (cm)	35.78 ± 3.9	35.65 ± 3.9	35.68 ± 3.9
Biceps skin fold (mm)	3.78 ± 0.7	3.59 ± 0.7	3.78 ± 0.6
Triceps skin fold (mm)	3.85 ± 0.6	3.68 ± 0.6	3.74 ± 0.6
Supra inguinal skin fold (mm)	5.41 ± 1.1	5.16 ± 1.1	5.29 ± 1.1
Sub-scapular skin fold (mm)	4.33 ± 1.5	4.16 ± 1.4	4.23 ± 1.4
Body composition
Body Fat (%)	37.9 ± 5.1	37.73 ± 4.3	37.18 ± 5.3
Visceral Fat (%)	17.87 ± 4.4	17.69 ± 4.5	17.44 ± 4.4
Subcutaneous Fat (%)	32.5 ± 9.1	32.5 ± 8.6	32.1 ± 9.1
Skeletal Muscle Mass (%)	23.9 ± 3.7	23.99 ± 3.6	24.2 ± 3.7
Vitals
Systolic Blood pressure (mmHg)	128 ± 6.5	126 ± 5.01	127.3 ± 7.9
Diastolic blood pressure (mmHg)	81.3 ± 5	79.33 ± 4.5	82 ± 4.14
Pulse (bpm)	76 ± 2	75 ± 1	75 ± 2

Fasting glucose levels showed a gradual reduction throughout the study. The difference between the glucose levels on days 0 and 45 was statistically significant. The fasting insulin levels increased immediately after the therapeutic course, which fell back to baseline levels on day 45. The lipid profile did not show any statistically or clinically significant change. Although leptin levels also showed a gradual decrease, no statistically significant change was noted (Table 2). The HOMA-IR was found to increase immediately after the enema course, which fell even below the baseline on day 45. The same trend was seen in the case of TG/HDL ratio (Table 3).

Table 2.

Effect of buttermilk administration on biochemical parameters

Parameter	Day 0	Day 16	Day 45
Fasting glucose mg %	94.4 ± 19.2	85.06 ± 12.7	79.2 ± 11.8*
Fasting Insulin (mU/L)	9.29 (0.76–34.34)	13.17 (5.28–54.79)	9.7 (1.24–21.86)
Leptin (ng/mL)	3.67 (0.70–5.84)	3.37 (0.87–6.31)	3.05 (0.41–6.22)
Lipid profile
Total Cholesterol mg %	175.8 ± 40.3	175.1 ± 30.0	182.7 ± 38.7
LDL mg %	126.1 ± 35.6	122 ± 25.08	132.8 ± 36.48
HDL mg %	36.3 ± 5.9	35.41 ± 5.1	38.31 ± 7.1
VLDL mg %	25.49 ± 10.6	28.66 ± 9.9	25.22 ± 7.6
Triglycerides mg %	127.4 ± 53.17	143.3 ± 49.68	126.1 ± 38.43

*p < 0.05 using Kruskal Wallis Test

Table 3.

Effect of buttermilk administration on insulin resistance

Parameter	Day 0	Day 16	Day 45
HOMA-IR	2.15 (0.15–8.31)	2.71 (0.94–13.93)	1.44 (0.28–5.13)
TG/HDL	3.51 (1.46–5.12)	4.11 (1.33–7.87)	3.39 (1.66–6.18)

Microbiota analysis for plain and processed buttermilk used for treatment

No significant batch to batch variation was observed in plain buttermilk used for preparation as well as after its processing with medicinal plants (p > 0.05). Lactobacillus and Streptococcus were observed to be the most dominating genera. Figure 1a and b compare the bacterial diversity at the genus level in plain and processed buttermilk samples, respectively.

Fig. 1.

Relative abundance of different genera in 3 different batches of plain buttermilk (a) & buttermilk preparation processed with medicinal plants (b)

Effect of buttermilk administration on faecal microbiota

To investigate differences among faecal microbiota composition of patients at 0th, 16th, and 45th day, alpha diversity analysis was performed by dividing samples based on sampling time. There was a difference in alpha diversity when faecal microbiota of day 0 was compared with day 16. On day 45, the faecal microbiota was almost similar to day 0. These changes were not significant statistically. (Fig. 2).

Fig. 2.

Alpha diversity analysis of stool at different time points by Chao1, Observed and Shannon Index

Effect of buttermilk administration on the relative abundance of different taxa

There was variation in abundances of major phyla observed at different time points. Firmicutes, Proteobacteria and Deinococcus-Thermus showed increase in abundances from 13.01%, 4.97% & 0.11% to 23.6%, 11.5% & 2.98%, respectively. Bacteroidetes abundance decreased from 80.72% to 61.08% on the 16th day (Fig. 3a). Firmicutes to Bacteroidetes ratio was found increased from day 0 (0.161) to day 16 (0.384), which returned to baseline level on day 45 (0.151).

Fig. 3.

Relative abundance of different bacterial taxa in stool samples at 3 different time points at Phylum level (a) & Genus level (b)

Stool samples also showed a change in bacterial diversity at genera level over three-time points (Fig. 3b). An increase in diversity was noted immediately after treatment (day 16), which got reduced after the follow up period (day 46). The microbial composition after the follow up period was not exactly similar to that observed at baseline. It showed the presence of certain genera and modified abundances. There was a shift towards high diversity and back to the relatively initial pattern over 45 days. The abundance plot (Fig. 4) clearly showed the emergence of genera Geobacillus, Meoithermus, Areibacillus, Culobacter etc. on day 16 and then subsequent decrease on day 45. Also, slight decrease in abundance of the genus Prevotella was observed which was restored to the original level on day 45.

Fig. 4.

Abundance of different genera at different time points depicting emergence of specific genera on day 16

Principal Coordinate Analysis (PCoA) plot based on the Bray-Curtis metric; showed significant overlap between day 0 and day 45 (Fig. 5). Day 16 samples showed distinct grouping as compared to day 0 and day 45 samples indicating differences in overall abundance and diversity just after the treatment. Moreover, the inter-individual diversity on day 16 was higher as compared to day 0 and day 45.

Fig. 5.

Beta diversity analysis. 3 clusters can be observed indicating change in the microbial diversity after treatment of buttermilk enema

There were changes noted in the abundances of few genera over time (Fig. 6). Genera Lactobacillus, Geobacillus, Pseudomonas showed an increase in abundance after buttermilk treatment (P < 0.05) which is again reduced on 45th day. Geobacillus showed a significant rise in all samples whereas earlier, they were negligible or absent in many samples. Faecalibacterium showed decreased abundance immediately after treatment (P = 0.05). Genus Megasphaera showed a significant and slight positive correlation (Fig. 7a) with fasting blood sugar levels of patients at both time points (Day 0 and 45) (P = 0.0187). Waist circumference showed significant positive correlation (P = 0.005) with BMI of all patients before treatment (Day 0) and at follow up (Day 45) (Fig. 7b).

Fig. 6.

Box plots depict change in abundance of different genera before treatment and after treatment

Fig. 7.

Correlation plots showing significant correlation between Fasting Sugar in patients and Megasphera abundance (a) & between Waist circumference and BMI in patients (b)

Functional prediction results

The Glycolysis/Gluconeogenesis, Butanoate metabolism and Insulin signalling pathways showed two-fold up regulation on day 16 which in turn decreased on day 45, but the decrease was not significant. Carbohydrate metabolism pathway was down-regulated on day 16. Rest of the pathways remained majorly unchanged throughout the treatment (Supplementary_fileS1).

Discussion

The present study evaluated effect of rectal administration of buttermilk processed with medicinal plants on anthropometry, biochemical parameters and gut microbiota in obese individuals. We observed no significant difference in weight and BMI of the patients throughout the study period. The circumferential measures reduced immediately after treatment. However, this reduction was not sustainable during follow-up period. A similar trend was observed in case of gut microbiota. The microbial diversity showed increased diversity immediately after therapeutic course, which was not maintained after follow up period. Interestingly, there was an increase in fasting insulin levels in all patients with a decrease in glucose levels indicating effect of buttermilk administration on insulin secretion. We did not observe any adverse effects during study over 45 days, indicating the safety of the procedure.

According to World Health Organization (WHO) fact sheet, approximately 39% of the global adult population is overweight or obese and prevalence has seen to be increasing worldwide. It has been established that gut microbiota plays a vital role in development of obesity and associated comorbidities. Microbes present in the gut are known to contribute for food absorption and low-grade inflammation therefore gut microbiome modulation is one of the ways to suppress the severity of inflammatory disorders like IBD, IBS as well as lifestyle disorders like type 1,2 diabetes, obesity and thus are considered potential sources of novel therapeutics [21].

Buttermilk is a known probiotic and used regularly as a part of the Indian diet [22, 23]. Milk fat globule membrane (MFGM) proteins in buttermilk are known to have cholesterol-lowering, antiviral, antibacterial, and anticancer properties [24]. In the present study, we used buttermilk processed with medicinal plants for the treatment in obese patients. This was a first systematic study to assess changes in anthropometric and metabolic parameters along with gut microbial composition in obese individuals after a therapeutic course of medicated buttermilk. We used buttermilk prepared in 3 different batches. The buttermilk samples from all 3 batches were initially tested to understand batch-to-batch variation in bacterial diversity among the samples and the changes occurring in buttermilk after processing it with medicinal plants. An increased bacterial diversity was noted in processed buttermilk samples compared to plain buttermilk samples indicating successful processing. As the preparation of buttermilk involves a crude procedure, some variation in microbial composition was seen amongst 3 batches. However, this variation did not affect its therapeutic effects to large extent. The microbiota analysis revealed more abundance of organisms like Geobacillus, Meoithermus, Aeribacillus, Culobacter etc. in processed buttermilk. Of these, Geobacillus, Meoithermus and Aeribacillus are known thermophilic, whereas genus Culobacter is commonly obtained in aquatic and soil environments. These findings indicate that plants used for processing buttermilk may be the source of these genera.

The weight and BMI though showed slight changes, both these parameters were almost constant throughout the study duration. Interestingly, circumferential measures and skinfold thicknesses showed reduction immediately after the therapeutic course of enema. The higher level of abdominal fat (higher abdominal girth) is associated with an increased risk of cardio-metabolic disease. Waist circumference (WC) provides a measure of fat distribution that cannot be obtained by measuring BMI. Therefore, WC is often used as a surrogate marker of abdominal fat mass also associated with cardio-metabolic disease risk [25]. The reduction in circumferential measures can be correlated with fat redistribution or lipolysis during treatment. Skinfold thickness has also been specified as sensitive predictor of obesity.

Fasting sugar level showed gradual decrease, which was statistically significant on day 45 while fasting insulin level increased immediately after the therapeutic course and got reduced after follow-up. Insulin resistance (HOMA-IR as well as TG/HDL ratio) showed slight increase immediately after the course but it decreased at the time of follow up. The increase in fasting insulin with gradual reduction in sugar level therefore can be considered as an indicator of increased insulin secretion. It is reported that whey glucagon-like peptide 1 (GLP-1) is responsible for stimulating insulin secretion, acts as anti-obesity agent and improves lean body mass [26]. The insulin secretory activity is enhanced by incretin pathway along with growth of some bifidobacteria & lactobacilli. It is possible that the buttermilk administration works through the same pathway and exerts insulin secretory activity [27].

It is reported that BMI is positively associated with blood pressure and also with the prevalence of hypertension [28]. In our study, systolic and diastolic blood pressure remained normal in all patients at three time points. Lipid profile is important in obese individuals because of its possible association with increased cardiovascular risk. We observed, no significant change in the lipid profile after the course of an enema and the follow up period. There was a gradual reduction in leptin, but it was not statistically significant. Leptin is a pro-inflammatory adipokine that plays an essential role in the regulation of energy metabolism. It also has a regulatory role in the interplay between energy metabolism and the immune system, responsible for the inflammation associated with obesity [29].

The change in the microbial diversity at phylum and genus levels was observed immediately after buttermilk treatment (day16) which was restored to the original state (day 0) on day 45. An increase in Firmicutes abundance and decrease in Bacteroidetes level was observed on day 16. The gut microbiota on 16th day had an influence of the treatment of buttermilk enema and thus such change in the microbial community structure could be a possible response to rectal administration of buttermilk. No substantial difference was observed in the Firmicutes to Bacteroidetes ratio between day 0 and day 45, though an increase in the ratio was observed as an immediate effect of buttermilk enema. Changes in abundances of Firmicutes and Bacteroidetes have been observed to have close association with obesity. An increased ratio of Firmicutes: Bacteroidetes has been reported in obese individuals compared to lean individuals. However, few studies have shown no trend or even the opposite trend in this ratio [30–32]. Alexander Koliada et al. have reported a higher level of firmicutes and lower level of bacteroidetes in obese individuals as compared to normal-weight and lean adults [33]. Whereas, Duncan et al. and Nadal et al. have observed no trend in terms of Firmicutes:Bacteroidetes ratio. Also, reports by Tims et al. and Schwiertz et al. State that they observed increased abundances of Bacteroidetes and reduced numbers of Firmicutes in obese individuals [30–32, 34]. Moreover, a report by Magne et al. found high variation for Firmicutes and Bacteroidetes ratio depending on population and stated that the association of this ratio to obesity is not persuasive [35].

Functional analysis by Piphillin revealed the upregulation of genes responsible for butanoate metabolism, suggesting increased butanoate synthesis in the colon. A report by Dallas R. Donohoe et al. states that colonocytes utilize bacterially-produced butyrate as their primary energy source [36]. Another report by Yael Litvak et al. showed that the alteration of metabolism of the colonic epithelium increases epithelial oxygenation and thus driving an expansion of facultative anaerobic bacteria, which is a hallmark of dysbiosis in the colon [37]. Carbohydrate metabolism remains unchanged throughout the treatment period and further got down-regulated. This result was further supported by decreased levels of fasting blood glucose.

Significant information is available correlating Traditional Chinese Medicine (TCM) with gut microbiome. A report states that the interplay between Herbal Medicines (HM) components and gut microbiota improves gut microbiota composition [38]. It further states that bio-transformations by gut microbiota leads to a change in bioavailability and toxicity of HM components; thus, stabilizing the microbiome. Another report shows the effect of TCM on gut microbiota, which in turn shows an effect on diseases associated with vital organs like the liver, stomach, heart and brain [39].

Ayurveda recommends the administration of buttermilk for managing obesity. There are few clinical studies wherein buttermilk has been administered through a rectal route for the treatment of gut disorders. Mallikarjun et al., discussed the use of buttermilk enema for the management of patients with Irritable Bowel Syndrome [13]. In the present study, we evaluated its effect on obesity, considering its probiotic potential. Probiotics, preparations containing live organisms, in adequate amounts are known to modulate gut microbiota and thus causing health benefits [23]. Our findings indicate the effects of buttermilk administration on the modulation of the gut microbiota of obese patients. However, the methodology needs standardization in terms of duration and frequency of administration for sustained effects.

The study also has a few additional limitations. As this was proof of concept study, we have used a small sample size, which was further heterogeneous concerning age, BMI, the glycaemic status of the patients etc. We have not followed a uniform dietary or activity plan in the patients; therefore, it becomes difficult to attribute the effect solely to buttermilk administration. Further, additional research is necessary to determine the role of medicinal plants in the process. According to Ayurvedic pharmacology, some of the plants have anti-obesity potential. We have observed some genera of plant origin in the processed buttermilk samples. A similar study using plain buttermilk can throw light on this aspect. Lastly, the microbiome data needs in-depth analysis to identify non-responders to buttermilk treatment in terms of modulation of the microbiota. This may help in identifying phenotypes ideal for buttermilk administration.

Conclusion

Our results suggest that per rectal administration buttermilk processed with medicinal plants alter the gut microbiota in obese individuals. However, the effects are sustained only for a specified period. The patients may have to undergo the same treatment repeatedly or for a longer duration to achieve sustained effects.

Author contributions

SM- Contributed for anthropometry assessment, coordination among all three study sites, management of data, manuscript drafting and revision.

KKD- Contributed for stool and buttermilk DNA extractions, microbiome data generation and analysis, manuscript drafting and revision.

NL- Contributed for Study drug preparation, patient recruitment and treatment procedure follow up of patients, clinical assessment.

PK- Contributed for Ethical approval and CTRI registration, counselling of participants and supervision of clinical part.

DD- Contributed for Supervision of conduct and data interpretation of microbiome study, manuscript revision.

YSS- Contributed for Critical inputs in planning the study, approval for microbiome study and analysis methodology, manuscript revision.

SB- Contributed for Study conception and design, data monitoring, data analysis and interpretation, manuscript revision.

Declarations

Institutional Ethics Committee BVDUCOA/EC/−3482/2017–18.

Clinical Trial Registry of India CTRI/2018/02/011859.

Conflict of interest

The authors declared that they have no conflict of interest.