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Microbiology and Biotechnology Letters

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Food Microbiology (FM)  |  Probiotics in Nutrition and Health

Microbiol. Biotechnol. Lett. 2022; 50(4): 477-487

https://doi.org/10.48022/mbl.2208.08008

Received: August 12, 2022; Revised: October 6, 2022; Accepted: October 11, 2022

Lactobacillus rhamnosus CBT-LR5 Improves Lipid Metabolism by Enhancing Vitamin Absorption

Dong-Jin Kim†, Tai Yeub Kim†, Yeo-Sang Yoon, Yongku Ryu, and Myung Jun Chung*

R&D Center, Cell Biotech, Co., Ltd., Gimpo 10003, Republic of Korea

Correspondence to :
Myung Jun Chung,      ceo@cellbiotech.com

Abstract

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Probiotics provide a symbiotic relationship and beneficial effects by balancing the human intestinal microbiota. The relationships between microbiota changes and various diseases may predict health abnormalities and diseases. Treatment with vitamins and probiotics is one therapeutic approach. To evaluate the effect of probiotics on vitamin absorption, we chose Lactobacillus rhamnosus CBT-LR5 treatment, which has resistance to vitamin C-inducible toxicity, with vitamins in high-fat diet (HFD)-induced obesity models. CBT-LR5 affected the absorption of micronutrients, such as ionic minerals and water-soluble vitamins. An increase in vitamin C absorption by CBT-LR5 enhanced the antioxidant response in HFD-induced obesity models. Increased vitamin B absorption by CBT-LR5 regulated lipid metabolism in HFD-induced obesity models. These favorable effects of CBT-LR5 on the absorption of vitamins should be investigated as candidate therapeutic target treatments for metabolic diseases.

Keywords: Probiotics, Lactobacillus rhamnosus, Vitamin C, Vitamin B, antioxidant response, lipid metabolism

Graphical Abstract


Introduction

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Probiotics are living microorganisms that exhibit a symbiotic relationship and balance the human intestinal microbiota [1, 2]. There are many beneficial effects of probiotics on human life [3]. Probiotics induce the bio-conversion of ingested food into an easily digested and absorbed form in the human intestine and inhibit the production of cholesterol and the growth of harmful bacteria or pathogens in the intestine that cause various diseases [1, 4]. Probiotics modulate the immune response by reducing inflammatory cytokines and activating intestinal epithelial cells, which improves the intestinal environment [5, 6].

Numerous studies investigated beneficial bacteria in the human gut, and some health abnormalities and diseases may be predicted based on changes in probiotics [7, 8]. Research progress identified the relationship between microbiota changes and intestinal diseases, including inflammatory bowel disease (IBD), colorectal cancer (CRC), and diarrheal diseases and their specific pathobionts [9, 10]. Recent studies confirmed correlations of microbiota with neuropsychiatric diseases, diabetes, obesity, and arteriosclerosis and suggested a clinical approach using probiotics to inhibit disease-specific pathobionts and change the environment and microbiota [11, 12].

Changes in the intestinal environment caused by probiotics are mediated by secretory organic acids, such as lactic acid [13]. Secretory organic acids produce a partially acidic environment that affects the absorption of minerals, including calcium and iron, by increasing their solubility [14, 15]. A recent study also reported that probiotics induced a higher absorption rate of vitamins and minerals from the gastrointestinal tract compared to milk or yogurt and increased the bioavailability of vitamins and minerals [16]. Therefore, taking probiotics with vitamins and minerals is a favorable approach for increasing the absorption efficiency of vitamins and minerals.

Vitamins are relevant for various metabolic processes in the body, including cell proliferation and death, and vitamins are essential nutrients for maintaining human health [17]. Vitamin deficiency weakens immunity and metabolism and increases the possibility of unexpected diseases [18−20]. Vitamins are classified as water-soluble and fat-soluble [21]. Most vitamins are obtained via food intake rather than synthesis in the body, and water-soluble vitamins, such as vitamin B and vitamin C, must be consumed continuously because of these agents are not stored in the body [22]. Vitamin B has a direct impact on mitochondrial function and toxicity and regulates energy metabolism as a coenzyme in diverse cell functions [23]. Vitamin C regulates collagen synthesis, energy metabolism, and tissue repair and functions as an antioxidant [24]. These properties of vitamins B and C are a promising approach to maintaining health and alleviating metabolic diseases.

Although probiotics play a role in synthesis of vitamins, the use of vitamins derived from probiotics is difficult in human body, because water-soluble vitamin is absorbed through the specific transporter in the duodenum and ileum, where probiotics are scarcely present [25−27]. Therefore, most of water-soluble vitamins are absorbed through external food, and many studies are focused on increasing the vitamin content in food using fermentation by probiotics [16, 28]. Recent studies showed that probiotics induced the increase of serum vitamin D and Lactobacillus reuteri NCIMB 30242 increased circulating 25-Hydroxyvitamin D in clinical trial and treatment with Saccharomyces cerevisiae and vitamin C ameliorated hyperglycemia, oxidative stress and dyslipidemia in alloxan-induced diabetic rats [29−31]. However, the relationship between probiotics and vitamin absorption was not clearly explained.

In this study, we demonstrated an increase in the intake of vitamins by probiotics and suggested the use of this combination in alleviating metabolic diseases. Furthermore, our study first described the relationship between lipid metabolism and probiotics-mediated vitamin absorption.

Materials and Methods

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Selection of probiotics

Bifidobacterium, Lactobacillus genus and Streptococcus were from Cell Biotech Co. Ltd. and their information was introduced our previous study [32]. The Bifidobacterium were cultured in a Bactron Anaerobic Chamber (Sheldon Manufacturing Inc., USA) using BL broth (Difco, USA) at 37℃ under anaerobic conditions (90% N2, 5% H2, 5% CO2). The Lactobacillus genus and Streptococcus were cultured aerobically at 37℃ using Lactobacilli MRS broth (Difco). To identify the survival rate against vitamin C of probiotic strains, Streptococcus thermophilus CBT-ST3, Lactobacillus acidophilus CBT-LA1, Lactobacillus rhamnosus CBT-LR5, Bifidobacterium longum CBT-BG7, Bifidobacterium bifidum CBT-BF3 and Bifidobacterium lactis CBT-BL3 were inoculated in 0, 0.12, 0.24, 0.36, 0.48 and 0.6 M vitamin C-containing MRS and BL broth for 16 h, and their CFUs were counted. For short-term toxicity against vitamin C, S. thermophilus CBT-ST3, Lactobacillus rhamnosus CBT-LR5, B. longum CBT-BG7 and B. lactis CBT-BL3, were inoculated for 20 min in MRS and BL broth (106−107 cfu/ml) containing 0, 0.03, 0.06, 0.15 and 0.3 M vitamin C. Samples were taken 20 min after inoculation and subjected to viable cell counts using a LIVE/DEAD BacLight kit™ (Invitrogen, USA) at 100x magnification under fluorescence microscopy BX50 (Olympus, Japan). Scale bar= 10 μm.

Animals

These studies were performed in 4-week-old rats (male SD rat, Saeron Bio, Korea). The animals had free access to irradiation-sterilized dry pellet-type feed and water during the study period. The breeding environment (specific pathogen-free facility) was maintained under the conditions of a temperature of 22 ± 2℃, relative humidity of 40 ± 20%, and a light-dark cycle of 12 h (Laboratory Animal Center of CellBiotech Co., Ltd., Korea). During the acclimatization period, the health of the rats was checked, and animals that did not show a decrease in activity were selected and used for subsequent experiments. In accordance with the study schedule, the rats were sacrificed by inhaling CO2 at the end of administration of the test substance. The animal use protocol was reviewed and approved by the Institutional Animal Care and Use Committee board in CellBiotech (IACUC, Approval No.: CBT-2016-14) based on the guidance of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC).

Oral administration of probiotics and vitamins (treatment with probiotics and vitamins)

To demonstrate the effect of probiotics on the absorption of vitamins, rats were randomized into four treatment groups (n = 6) and fed a high-fat diet (HFD, 60% of calories from fats) until the end of the experiment. The animals received oral treatments of 0.9% saline (control I), 5×109 cfu/head probiotics (L. rhamnosus CBT-LR5, treat I), multivitamin mixture (treat II, vitamin B1: 1.2 mg, B2: 1.4 mg, B3: 15.02 mg, B6: 1.5 mg, A: 700.24 μg, E: 11.02 mg, C: 100.12 mg, D: 5 μg/Head), and a combination of probiotics and multivitamin mixture (treat III) (Table 1). Each treatment was continued for five weeks with a regimen of five times per week.

Table 1 . Oral-administration of CBT-LR5 and multivitamin.

GroupTreatmentCondition
G1 (NC)HFD + PBSNon
G2 (LR)HFD + CBT-LR5CBT-LR5 : 5 × 109 CFU/Head
G3 (M.Vit)HFD + MultivitaminA: 700.24 ug/Head
E: 11.02 mg/Head
C: 100.12 mg/Head
D: 5 ug/Head
B1: 1.2 mg/Head
B2: 1.4 mg/Head
B3: 15.02 mg/Head
B6: 1.5 mg/Head
G4 (LR+M.Vit)HFD + CBT-LR5 + MultivitaminCBT-LR5 : 5× 109 CFU/Head + Multivitamin
Multivitamin: vitamin A, vitamin E, vitamin C, vitamin D, vitamin B plex (B1, B2, B3, B6)


To verify the disease inhibition effects of enhanced vitamin absorption according to probiotics, we performed two independent experiments. First, the rats were randomized into four treatment groups (n = 6) and fed HFD until the end of the experiment: 0.9% saline (control I), 5 × 109 cfu/head probiotics (L. rhamnosus CBT-LR5, treat I), 1000 mg/head L-ascorbic acid (treat II), and a combination of probiotics and L-ascorbic acid (treat III) (Table 2). Each treatment was continued for five weeks with a regimen of five times per week.

Table 2 . Oral-administration of CBT-LR5 and vitamin C.

GroupTreatmentCondition
G1 (NC)HFD + PBSNon
G2 (LR)HFD + CBT-LR5CBT-LR5: 5 × 109 CFU/Head
G3 (Vit.C)HFD + L-ascorbic acidVitamin C: 1000 mg/Head
G4 (LR+Vit.C)HFD + CBT-LR5 + Vitamin CCBT-LR5: 5 × 109 CFU/Head
Vitamin C: 1000 mg/Head


To study the sickness-inhibiting effect due to enhanced vitamin absorption with the probiotics, rats were randomly allocated to four treatment groups (n = 6) and fed HFD to the end of the experiment. Oral doses of 0.9 percent saline (control I), 5 × 109 cfu/head of probiotics (L. rhamnosus CBT-LR5, treat I), vitamin B complex (vitamin B1: 18 mg/head, B2: 21 mg/head, B6: 22.5 mg/ head, B7: 0.045 mg/head, treat II), and a combination of probiotics and vitamin B complex (treat III) were administered from day one (Table 3). Each therapy was given five times each week for a total of five weeks.

Table 3 . Oral-administration of CBT-LR5 and vitamin B complex.

GroupTreatmentCondition
G1 (NC)HFD + PBSNon
G2 (LR)HFD + CBT-LR5CBT-LR5 : 5 × 109 CFU/Head
G3 (Vit.B)HFD + Vitamin B complexB1: 18 mg/Head
B2: 21 mg/Head
B6: 22.5 mg/Head
B7: 450 ug/Head
G4 (LR+Vit.B)HFD + CBT-LR5 + Vitamin B complexCBT-LR5 : 5 × 109 CFU/Head + Vitamin B complex
Vitamin B complex : Thiamine Hydrochloride (B1), Riboflavin (B2), Pyridoxine Hydrochloride (B6), Biotin (B7)


Biochemical parameters

The animals were fasted for 16 h and sacrificed using inhalational anesthesia (isoflurane) at 5 weeks after treatment. Plasma was isolated from the blood and stored at -70℃ for use in biochemical analyses after centrifugation at 3000 rpm for 15 min. Blood was collected in tubes with anticoagulants (Vacuplast® collection line). Iron, calcium, total antioxidant status (TAS), superoxide dismutase (SOD), total cholesterol (CHOL), triglycerides (TGs), high-density lipoprotein cholesterol (HDL) and low-density lipoprotein cholesterol (LDL) were quantified in plasma using an enzymatic colorimetric assay (Erba Diagnostics Mannheim GmbH, Germany). Vitamins A, C, E, B1 and B6 were quantified in plasma using HPLC (high-performance liquid chromatography) with the respective detection kits. Vitamin B2 was quantified in plasma using a radioimmunoassay (1,25-dihydroxy vitamin D 123-I RIA).

Assessment of body weight and feed intake

During the course of the study, body weight, water and diet intake were weekly measured at a set time using an electronic balance for five weeks. To calculate feed efficiency ratio, the total amount of weight gain during the study period was divided by the total amount of feed consumption during the same time [33].

Detection of lactic acid bacteria changes in the gut

The effects of the oral administration of probiotic and vitamin combinations on gut microbial ecology were examined. The instructions on the MP Bio Stool Kit were followed for DNA preparation. The amount of lactic acid bacteria was calculated, and the values were compared using a real-time PCR assay with a primer specific for L. rhamnosus (Forward: TTG GGC GGT ATT TTA GCC GT, Reverse: TTG GTG AAG AAG CGG GTG TT).

Statistical analysis

The animal study data were statistically analyzed using Prism Version 6.0, and the data results were presented as the means and standard deviation (mean ± SD). Animal study data were evaluated using one-way ANOVA followed by Tukey's multiple comparison posttest when significance was observed. A value of p < 0.05 was considered statistically significant.

Results

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Lactobacillus rhamnosus CBT-LR5 resists vitamin C-induced toxicity

The antioxidant effect of vitamin C directly affects the growth inhibition and death of probiotics [34]. Therefore, it is necessary to select probiotics that are resistant to vitamin C before evaluating the efficacy. First, various concentrations of vitamin C were combined with 6 species of probiotics, S. thermophilus CBT-ST3, L. acidophilus CBT-LA1, L. rhamnosus CBT-LR5, B. longum CBTBG7, B. bifidum CBT-BF3 and B. lactis CBT-BL3, for 16 h to confirm the survival rate of the probiotics. Most probiotics, except L. rhamnosus CBT-LR5, died in media containing 0.12 M vitamin C. However, L. rhamnosus CBT-LR5 showed a 50% survival rate in media containing 0.12 M vitamin C. Although L. rhamnosus CBT-LR5 died in media containing 0.24 M vitamin C, L. rhamnosus CBT-LR5 had resistance to vitamin C compared to the other probiotics (Fig. 1A).

Figure 1.Selection of resistant probiotics against vitamin C inducible toxicity. (A) Survival rate of Streptococcus thermophilus CBTST3, Lactobacillus acidophilus CBT-LA1, Lactobacillus rhamnosus CBT-LR5, Bifidobacterium longum CBT-BG7, Bifidobacterium bifidum CBT-BF3 and Bifidobacterium lactis CBT-BL3 in various concentration of vitamin C-containing MRS and BL broth at 16 h after treatment. (B) The fluorescence images at 100x magnification under fluorescence microscopy for short-term toxicity of S. thermophilus CBT-ST3, L. rhamnosus CBT-LR5, B. longum CBT-BG7, and B. lactis CBT-BL3 in various concentrations of vitamin C-containing MRS and BL broth at 20 min after treatment. Scale bar = 10 μm.

To confirm the short-term toxicity of vitamin C on probiotics, 4 species of probiotics, S. thermophilus CBTST3, L. rhamnosus CBT-LR5, B. longum CBT-BG7, and B. lactis CBT-BL3, were cultured in media containing various concentrations of vitamin C for 20 min. Fluorescence was measured using a BacLight kit to identify the number of live probiotics. B. longum CBT-BG7 started to die in media containing 0.03 M vitamin C. S. thermophilus CBT-ST3 and L. rhamnosus CBT-LR5 died in media containing 0.3 M vitamin C. However, B. lactis CBT-BL3 had no short-term toxicity to vitamin C (Fig. 1B). Although L. rhamnosus CBT-LR5 showed short-term toxicity to vitamin C, it was selected for subsequent studies because it showed a high survival rate against long-term exposure to vitamin C.

Lactobacillus rhamnosus CBT-LR5 enhances the absorption of water-soluble micronutrients

In normal conditions, the levels of blood cholesterol and triglyceride are not enough to show the effect on the regulation of lipid metabolism by materials that reduce the blood cholesterol and triglyceride in high-fat diet mice [35−37], we experimented only in HFD-induced obesity model. To demonstrate the effect of CBT-LR5 on the absorption of vitamins and minerals, we administered CBT-LR5 with or without a multivitamin complex, which included vitamins A, C, E, D, and 4 types of vitamin B, in an HFD-induced obesity model (Table 1). Five weeks after treatment, the body weights and FER were not significantly different between the groups (Figs. 2A and B). Multivitamin treatment increased the absorption of iron and it was enhanced by multivitamins with CBT-LR5 treatment (Fig. 2C). Calcium absorption was increased by multivitamins with CBT-LR5 treatment (Fig. 2D). Multivitamin treatment increased the levels of vitamins in serum. Notably, multivitamin treatment with CBT-LR5 enhanced the absorption of vitamin C. However, CBT-LR5 did not enhance the absorption of vitamin A and E (Figs. 2E−G). Multivitamin treatment decreased the levels of total cholesterol. Total cholesterol was lower in multivitamin with CBT-LR5 treatment than multivitamin without CBT-LR5 treatment (Fig. 2H). However, levels of triglycerides, LDL and HDL were not significantly different between the groups (Figs. 2I−K). The change in the amount of intestinal CBT-LR5 was confirmed using fecal analysis, which confirmed that the amount of LR was increased in the feces of the CBT-LR5 treatment groups. The simultaneous administration with multivitamin did not reduce the stability of CBT-LR5 (Fig. S1A). Although the absorption of vitamin B was not identified due to their low concentration in this model, these results suggest that CBTLR5 increases the absorption of water-soluble micronutrients, such as vitamin C and ionic minerals.

Figure 2.The effect of multivitamins with Lactobacillus rhamnosus CBT-LR5 on the absorption of water-soluble micronutrients in HFD-induced obesity model. (A) The change of body weight during experiment. (B) The change of food efficiency ratio (FER) during experiment. (C) The grape of blood iron level at 5 weeks after treatment. (D) The grape of blood calcium level at 5 weeks after treatment. (E) The grape of blood vitamin C level at 5 weeks after treatment. (F) The grape of blood vitamin A level at 5 weeks after treatment. (G) The grape of blood vitamin E level at 5 weeks after treatment. (H) The grape of blood total cholesterol level at 5 weeks after treatment. (I) The grape of blood triglycerides level in blood serum at 5 weeks after treatment. (J) The grape of LDL level at 5 weeks after treatment. (K) The grape of blood HDL level at 5 weeks after treatment. Values are shown as the mean ± S.E.M. *p < 0.05 versus NC, #p < 0.05 versus LR, †p < 0.05 between two groups.

Lactobacillus rhamnosus CBT-LR5 increases the antioxidant effect of vitamin C

To confirm the effect of CBT-LR5 on the absorption of water-soluble vitamins, we performed in vivo experiments for each water-soluble vitamin separately. Vitamin C was administered with or without CBT-LR5 in the HFD-induced obesity model. Five weeks after treatment, vitamin C inhibited body weight gain. However, vitamin C with CBT-LR5 treatment did not cause additional body weight loss compared to vitamin C without CBT-LR5 treatment (Fig. 3A). FER was not significantly different between groups (Fig. 3B). Vitamin C treatment increased the level of vitamin C in blood, and its level was higher in the vitamin C with CBT-LR5 treatment than the vitamin C without CBT-LR5 treatment group (Fig. 3C). Because vitamin C is an antioxidant, we measured the total antioxidant status (TAS) and levels of superoxide dismutase (SOD) in blood using ELISA. Treatment with vitamin C increased TAS and SOD, and their induction was higher in the vitamin C treatment with CBT-LR5 than the vitamin C treatment without CBT-LR5 (Figs. 3D and E). Fecal analysis confirmed that the amount of LR was increased in the feces of the CBT-LR5 treatment groups, and simultaneous administration with vitamin C did not reduce the stability of CBT-LR5 (Supplementary Fig. S1B). These results suggest that CBT-LR5 increases the absorption of vitamin C, and the increase in vitamin C by CBT-LR5 enhances the antioxidative response in the body.

Figure 3.The effect of increased vitamin C absorption by Lactobacillus rhamnosus CBT-LR5 on the antioxidant response in HFD-induced obesity model. (A) The change of body weight during experiment. (B) The change of food efficiency ratio (FER) during experiment. (C) The grape of blood vitamin C level at 5 weeks after treatment. (D) The grape of blood total antioxidant status (TAS) level at 5 weeks after treatment. (E) The grape of blood superoxide dismutases (SOD) level at 5 weeks after treatment. Values are shown as the mean±S.E.M. *p < 0.05 versus NC, #p < 0.05 versus LR, †p < 0.05 between two groups.

Lactobacillus rhamnosus CBT-LR5 regulates lipid metabolism via the vitamin B complex

Since the absorption of vitamin B was not identified in previous experiment, the concentration of vitamin B complex was increased and administered with or without CBT-LR5 in the HFD-induced obesity model to confirm the enhancement of vitamin absorption. Five weeks after treatment, the gain of body weight was inhibited by the vitamin B complex. However, vitamin B complex with CBT-LR5 treatment did not cause additional body weight loss compared to vitamin B complex without CBT-LR5 treatment (Fig. 4A). FER was not significantly different between groups (Fig. 4B). Vitamin B complex treatment increased the levels of vitamin B1, B2 and B6, and their levels were higher in the vitamin B complex with CBT-LR5 treatment group than the vitamin B without CBT-LR5 treatment group (Figs. 4C−E). Vitamin B treatment decreased the levels of total cholesterol, LDL and triglycerides and increased the level of HDL in blood (Figs. 4F−I). Total cholesterol and triglycerides were lower in vitamin B with CBT-LR5 treatment than vitamin B without CBT-LR5 treatment (Figs. 4F and G). The atherogenic index of blood, which is a strong marker to predict cardiovascular disease, was significantly decreased by vitamin B with CBT-LR5 treatment (Fig. 4J). The amount of LR was increased in the feces of the CBT-LR5 treatment groups, and simultaneous administration with the vitamin B complex did not reduce the stability of CBT-LR5 (Fig. S1C). These results indicate that LR increases the absorption of the vitamin B complex and vitamin B-mediated additional benefit to lipid metabolism.

Figure 4.The effect of increased vitamin B absorption by Lactobacillus rhamnosus CBT-LR5 on lipid metabolism in HFD-induced obesity model. (A) The change of body weight during experiment. (B) The change of food efficiency ratio (FER) during experiment. (C) The grape of blood vitamin B1 level at 5 weeks after treatment. (D) The grape of blood vitamin B2 level at 5 weeks after treatment. (E) The grape of blood vitamin B6 level at 5 weeks after treatment. (F) The grape of blood total cholesterol level at 5 weeks after treatment. (G) The grape of blood triglycerides level at 5 weeks after treatment. (H) The grape of LDL level at 5 weeks after treatment. (I) The grape of blood HDL level at 5 weeks after treatment. (J) The grape of atherogenic index at 5 weeks after treatment. Values are shown as the mean ± S.E.M. *p < 0.05 versus NC, #p < 0.05 versus LR, †p < 0.05 between two groups.

Discussion

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The most commonly used probiotics to ameliorate disease are Lactobacillus and Bifidobacterium species, and their beneficial effects inhibit the growth of pathobionts and modulate the immune system. L. rhamnosus (LR) has been extensively studied in relation to various diseases, and clinical approaches to treating diseases using these functions are under investigation. The L. rhamnosus strain HN001 improved the absorption of minerals, enhanced the growth of bone and reduced the risk of postpartum anxiety and postnatal depression [38, 39]. Another stain, L. rhamnosus 1.0320, regulated the immune system by increasing splenic lymphocyte proliferation, and L. rhamnosus LDTM 7511 had an anti-inflammatory effect in DSS-induced colitis mice via gut microbiota modulation [40, 41]. Our own strain of L. rhamnosus, CBT-LR5, produces anticancer protein P8, which suppresses CRC proliferation and regulates RNF152 expression to induce the apoptosis of CRC [42, 43]. We confirmed that CBT-LR5 was resistant to vitamin C for long-term toxicity compared to other Lactobacillus and Bifidobacterium species.

Recent studies suggest that the coadministration of vitamins and L. rhamnosus promotes health [44]. L. rhamnosus GG (LGG) with vitamin D improved the immunological effectiveness of grass-specific sublingual immunotherapy in children with allergies, and the combination of LGG, vitamin D and zinc prevented atopic dermatitis in infancy [45, 46]. However, the effect of L. rhamnosus and water-soluble vitamins on disease has not been well studied. The combination of cranberries, L. rhamnosus and vitamin C had a beneficial effect in the management of urinary tract infection, and the combination of B. longum BB536 and L. rhamnosus HN001 with vitamin B6 improved the symptoms and severity of irritable bowel syndrome by restoring intestinal permeability and gut microbiota [47−49]. However, these effects were due to the function of L. rhamnosus itself and were not related to the efficacy of vitamin absorption. The present study focused on the efficacy of vitamins with increased absorption from CBT-LR5 and demonstrated improvement of water-soluble vitamin and ionic mineral absorption in an HFD-induced metabolic disease model. As a result of CBT-LR5-improved vitamin C absorption, TAS and SOD, markers of the antioxidant response, were increased, and the CBT-LR5improved vitamin B absorption modulated lipid metabolism with a reduction in total cholesterol and triglycerides in the HFD-induced metabolic disease model. CBT-LR5 maintained intestinal stability when treated with vitamin despite the in vitro vitamin-induced toxicity. Taken together, CBT-LR5 with vitamins increased vitamin absorption, and these changes enhanced vita-min-mediated effects, including improvements in the antioxidant response and lipid metabolism. These results show the possibility of overcoming metabolic diseases.

A diverse strain of L. rhamnosus has known as vitamin B-producing probiotics through the fermentation of food [50−52]. L. rhamnosus F5 can produce vitamin B12 via biofortification of soymilk and L. rhamnosus LC705 from cheese owns a gene for the production of B1, B6, and B9 [50, 51]. Furthermore, L. rhamnosus CRL 1963 produces vitamin B2 and L. rhamnosus CRL 1972 produces vitamin B9 from the degradation of phytate in quinoa sourdough [52]. However, LGG and L. rhamnosus yuba catabolites and degrades vitamin C for its stability and their growth during fermentation in milk and fruit jam [53−55]. Therefore, further studies will be needed to demonstrate whether CBT-LR5 produces vitamins and thereby increases adsorption.

The probiotic L. rhamnosus grows in a symbiotic relationship in the host intestine and regulates the pH in the digestive tract by secreting organic acids, such as lactic acid, to produce acidic conditions [56]. However, when the intestinal flora changes and the distribution of lactic acid bacteria, such as L. rhamnosus, decreases, pH imbalance occurs in the digestive system [57]. Changes in intestinal pH in patients with achlorhydria or long-term use of antacids lower the absorption capacity of vitamins and causes calcium and iron deficiency [58]. Intake a phytate-rich meal with L. plantarum 299v produces the lactic acid by phytase activation and lactic acid lowers pH, forms soluble iron complexes and finally increases iron absorption [59, 60]. Furthermore, some lactic acid producing probiotics with high phytate meals influences iron absorption and iron status markers [61]. As a similar example, the change in intestinal pH caused by L. rhamnosus may explain our result that CBT-LR5 increased the absorption of vitamin B, vitamin C, and minerals.

In conclusion, we demonstrated that CBT-LR5 improved the absorption of vitamin C, vitamin B and minerals, and theses increase induced an antioxidative effect and regulated lipid metabolism. These favorable effects of CBT-LR5 on the absorption of micronutrients should be investigated as a candidate therapeutic target treatment for metabolic diseases. However, there are some limitations in present study. 1) We have not checked whether other strain of L. rhamnosus also increase the absorption of water-soluble vitamins. 2) The change in intestinal pH was not clearly explained in this study. Therefore, further studies on the induction of acidic conditions by probiotics and their secreted organic acids and comparative studies on vitamin absorption between L. rhamnosus strains are needed.

Conflict of Interest

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The authors have no financial conflicts of interest to declare.

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