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Food Microbiology (FM) | Probiotics in Nutrition and Health
Microbiol. Biotechnol. Lett. 2022; 50(4): 477-487
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, firstname.lastname@example.org
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
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 . 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 . 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 . 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 . Vitamin deficiency weakens immunity and metabolism and increases the possibility of unexpected diseases [18−20]. Vitamins are classified as water-soluble and fat-soluble . 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 . Vitamin B has a direct impact on mitochondrial function and toxicity and regulates energy metabolism as a coenzyme in diverse cell functions . Vitamin C regulates collagen synthesis, energy metabolism, and tissue repair and functions as an antioxidant . 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
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.
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).
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 (
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 (
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 (
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).
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 .
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
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
The antioxidant effect of vitamin C directly affects the growth inhibition and death of probiotics . 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,
To confirm the short-term toxicity of vitamin C on probiotics, 4 species of probiotics,
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.
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.
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.
The most commonly used probiotics to ameliorate disease are
Recent studies suggest that the coadministration of vitamins and
A diverse strain of
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
This research was supported by Cell Biotech, Co., Ltd., Korea.
The authors have no financial conflicts of interest to declare.
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