CN112930392B - Probiotics for upper respiratory tract infections, stress, anxiety, memory and cognitive dysfunction and aging - Google Patents
Probiotics for upper respiratory tract infections, stress, anxiety, memory and cognitive dysfunction and aging Download PDFInfo
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Abstract
The first aspect of the invention relates to a strain Lactobacillus plantarum (lactobacillus plantarum) which is preserved in China general microbiological culture collection center (CGMCC), the preservation number is CGMCC15535, and the whole genome sequence is uploaded in GenBank accession number is CP031318. The strain has unique beneficial functions on upper respiratory tract infection, stress, anxiety, memory and cognition dysfunction, alzheimer disease, hyperlipidemia, intestinal homeostasis and aging.
Description
Technical Field
This application claims the full benefit of the malaysia patent PI2018703091 submitted on the date of delivery of 2018, 9, 3, and the entire contents of the patent document submitted in malaysia are incorporated herein by reference and referred to herein as my priority application.
The field of the invention is medicine, microbiology and nutrition, in particular to probiotics Lactobacillus plantarum DR. The strain has a series of beneficial biological functions, is beneficial to health, and has effects of preventing and treating upper respiratory tract infection, stress, anxiety, memory and cognitive dysfunction, and delaying aging.
Background
Scientific papers entitled "Lactobacillus plantarum DR" reducing cholesterol by phosphorylating AMPK to down regulate HMG-CoA reductase mRNA expression "(Lactobacillus plantarum DR7 reduces cholesterol via phosphorylation of AMPK that down-regulated the mRNA expression of HMG-CoA reductase) have been published in 2018, 4, 30. The invention referred to in this paper is the same as in this patent, but published for a period of one year before the malaysia filed patent. The content of the paper is incorporated into my priority application (so that the paper content is not published prior to the priority application) and also appears in this application. The authors of the articles are the inventors of the present patent, all signing the rights involved in the present patent and my priority application; in other words, the paper can be regarded as "original disclosure of the inventor" (the main material of the disclosure must be the inventor's own or the joint inventor, or directly granted by the inventor and the joint inventor). The paper describes for the first time the underlying mechanism of Lactobacillus plantarum DR7 in cholesterol metabolism. According to the discussion above, lactobacillus plantarum DR7 and its medical applications, as a patentable subject, should be within a annual grace period within a country.
Paper "Lactobacillus plantarum DR reduces stress and anxiety via serotonin and dopamine pathways, improving memory and cognitive ability in stressed adults" (Lactobacillus plantarum DR, alleviated stress and anxiety while improving memory and cognition in stressed adults via the serotonin and dopamine pathways) was published in month 3, 18, 2019, later than the priority date of my prior application.
The content of the above paper is incorporated into my priority application (so that the content is not published prior to application submission) and is also incorporated into this application. The Lactobacillus plantarum DR strain was therefore unpublished prior to the submission of my priority application in improving memory and cognition, and in relieving stress and anxiety.
Paper "Lactobacillus plantarum DR" improves upper respiratory tract infections by enhancing immune and inflammatory parameters a randomized, double-blind, placebo-controlled study "(Lactobacillus plantarum DR, improved upper respiratory tract infections via enhancing immune and inflammatory parameters: A r and ombed, double-blind, placard-controlled study) was published on month 4 of 2019 for 3 days later than the priority date set forth in my priority application. The content of the above paper is incorporated into my priority application (so that the content is not published prior to application submission) and is also incorporated into this application. The Lactobacillus plantarum DR strain was therefore unpublished prior to the filing of my priority application in improving upper respiratory tract infection inflammation and enhancing immunity.
The above-mentioned papers are incorporated herein in their entirety.
According to (FAO\WHO, 2006), probiotics are defined as "living microorganisms," and intake of certain amounts is beneficial to human health. The lactobacillus has health-care effects in improving intestinal microecology, regulating metabolic disorder, regulating immune response and other directions.
In dry seasons, pollutants suspended in the air excite inflammatory reactions through the gas, resulting in increased permeability of mucous membranes and increased susceptibility to pathogenic bacteria. In rainy season, the moist air increases the growth and reproduction of fungi such as mould and viruses, which leads to the easily-caused diseases of airborne infection such as cough and allergy. Upper respiratory tract infections are a general term for upper respiratory tract multiple infections, including common colds, laryngitis, pharyngitis, sinusitis. Is a representative disease of global health threat. In the united states, adults develop about 2-3 upper respiratory tract infections per year, while children do so as often as 5 times. Furthermore, more than 20% of adults have experienced an upper respiratory tract infection every 4 weeks.
Alzheimer's Disease (AD) is a globally prevalent dementia, with more than 4700 thousands of patients, consuming up to $6040 billion worldwide for this disease in 2018. 12% (approaching 8 billion) of the world's population is at age 60 years and older, AD has become an epidemic disease, and there is no effective therapeutic. The disease is characterized by the appearance of amyloid plaques, with degradation of, for example, amyloid beta (aβ). Enrichment of such polypeptides accelerates and doubles the conversion to insoluble fibers.
Stress is the non-specific reaction of human body under stress, which causes anxiety, discomfort, emotional tension and is difficult to regulate. Anxiety is the first physiological response to stress and prolonged anxiety can lead to mental disorders such as depression. Over 3 million people worldwide are affected by depression, with nearly 80 tens of thousands of people suicidal for the disease each year. The intestinal flora and brain health are closely interacted by the connection of intestinal brain axes, and bidirectional regulation is presented.
Aging is based on an age-related multifactorial process, associated with physiological degradation is an increase in age-related mortality. Aging is inevitable, so that current researchers are oriented to improve quality of life and life expectancy.
The effects of the aging process on the brain are multipath from the cellular functional hierarchy affecting perception, movement, and high level cognition. Some cognitive degenerations are associated with neurodegenerative diseases, such as Alzheimer's disease and memory degeneration. At the same time, aging is also associated with metabolic system diseases, such as hyperlipidemia, increasing the risk of cardiovascular and cerebrovascular diseases. The world health organization predicts that 31% of the global mortality is due to side effects of cardiovascular and cerebrovascular diseases. Thus, natural diet strategies can have positive practical implications in aging and heart brain tube disease. Aging affects physical indicators of the human body, such as the endurance of the body. Aging affects the molecular and cellular levels of the body, and thus the resulting effects are systemic to the body. For example, the physiological function of body muscles is deteriorated by aging. These phenomena can also be ameliorated and alleviated by some complex movements. In addition, molecules produced by cellular respiration have damage to cells, which in turn stimulates the body to accelerate the progression of aging. These molecules may be converted to oxidized proteins or other cellular molecules. Aging is a major causative agent in degenerative diseases of the brain such as Alzheimer's disease.
The length of telomeres is also commonly used as a biomarker for aging and age. Telomeres are nuclear protein complexes at the end of eukaryotic chromosomes that decay after each cell division. Each cell division causes a decrease in telomere length until a critical length is reached, initiating senescence of the cells.
When the body functions of the aged are much dysfunctional compared with the previous young period, the resulting phenomenon of discrimination against senile is more aggravated by the physical and mental deterioration of the aged. For example, it is difficult for glands to produce enough hormones with specific activities to affect the body's inability to perform biochemical processes normally. As a more visual example, women fail to produce sufficient sex hormone during menopause due to atrophy of the uterus and ovaries, and eventually fail to conception. In addition, aging can also lead to decreased hearing and vision, sleep disorders, muscle and calcium loss. Hormonal changes caused by aging also lead to skin health deterioration, and wrinkles and spots appear.
The biochemical reactions inside the organism and the organism metabolism are highly influenced by age factors. Biochemical reactions and body metabolism can be regulated by adenylate phosphoprotein kinase (AMPK). AMPK is an energy sensor inside cells, participates in synthesis and decomposition pathways, and plays an important role in balance control in cellular energy metabolism. When AMPK monitors a decrease in intracellular ATP levels, the stimulation of the relevant pathways rapidly stimulates energy metabolism to produce enough ATP for fatty acid oxidation and autophagy. Alternatively, AMPK also regulates transcription by means of phosphorylation of enzymes, which down-regulate ATP-consuming biological reaction processes, e.g. synthesis of glycogen, phospholipids, proteins, phosphorylation of transcription factors, co-expression coactivation, etc. Thus, AMPK regulates ATP levels to control the function of the body, ensuring that the body is maintained in an optimal condition, is a biological switch for controlling metabolism and aging. Recently, it has also been studied that AMPK activates macrophages upon the occurrence of infectious diseases through phosphorylation, showing that it also plays an important role in immune function and against viral infection.
Hyperlipidemia may be defined generally by elevated cholesterol and triglycerides. Hyperlipidemia is an important causative agent of cardiovascular and cerebrovascular diseases accompanied by elevated cholesterol and triglycerides. Hyperlipidemia can affect the antioxidant status of organs and the levels of phospholipidproteins, thereby causing metabolic abnormalities, increasing the incidence of diseases such as cardiovascular and cerebrovascular diseases and nonalcoholic liver injury. Probiotics are found in animal experiments to improve the health of phospholipid levels in blood.
The disorder of intestinal flora can cause problems such as diseases, appetite decrease, aging acceleration, etc. Aging and high fat diets are recognized as important factors affecting the state of the flora. Intestinal flora is a general term for all microorganisms in the intestinal tract, which forms a complex metabolic network, playing an important synergistic role in the metabolism of the host. It is hypothesized that by modifying the structure of the intestinal flora, the flora metabolism can be altered, thereby affecting the host's metabolism. Recent studies have increasingly revealed an important link between intestinal flora and metabolism in terms of host energy metabolism, metabolic diseases, bone health, etc.
In summary, the improvement of the above diseases continues to be an effective treatment regimen, especially for effective probiotic strains.
DISCLOSURE OF THE INVENTION
Technical problem to be solved
The key problem to be solved by the present invention is to provide new compositions and solutions based on the above mentioned conditions, which safely and effectively alleviate the above mentioned health problems.
Solution scheme
The solution is based on providing a probiotic strain Lactobacillus plantarum with a collection number of CGMCC15535, also called Lactobacillus plantarum DR7. The inventors have found that the relevant biological functions of the strain contribute to the improvement of different symptoms and diseases, in particular diseases as will be elaborated on below.
The first aspect of the invention relates to Lactobacillus plantarum strain or metabolite thereof, wherein the strain is preserved in China general microbiological culture collection center (CGMCC), the preservation number is CGMCC15535, the whole genome sequence is uploaded in GenBank accession number is CP031318, and the metabolite comprises 2-hydroxyisohexanoic acid and 3-phenyllactic acid.
One aspect of the invention relates to the Lactobacillus plantarum DR strain or metabolite thereof for use in therapy or as a medicament. This aspect may also be described as a method of probiotic treatment comprising administering an effective amount of Lactobacillus plantarum DR7 or a metabolite thereof, as desired. As used herein, the term "probiotic" refers to an active microorganism that, when administered in sufficient quantity, is beneficial to host health.
Another aspect of the invention relates to the use of strain Lactobacillus plantarum DR7 or a metabolite thereof for the prevention and treatment of upper respiratory tract infections by exerting anti-inflammatory and immunomodulatory protection.
Another aspect of the invention relates to strain Lactobacillus plantarum DR7 or a metabolite thereof for use in the treatment and prevention of stress, anxiety, memory and cognitive dysfunction by promoting, for example, the 5-hydroxytryptamine, dopamine pathways.
Another aspect relates to the use of strain Lactobacillus plantarum DR7 or a metabolite thereof for the prevention and treatment of neurodegenerative diseases.
In another aspect, the invention relates to strain Lactobacillus plantarum DR7 or a metabolite thereof to slow signs of aging by preventing telomere shortening and/or enhancing energy metabolism.
Another aspect of the invention relates to the prevention and treatment of hyperlipidemia and liver fat accumulation by inhibiting elevated triglyceride levels, inflammation, and fat accumulation by strain Lactobacillus plantarum DR7 or its metabolites.
Another aspect of the invention relates to strain Lactobacillus plantarum DR7 or a metabolite thereof for use in the prevention and treatment of cardiovascular disease and for cholesterol reduction.
Another aspect of the invention relates to strain Lactobacillus plantarum DR7 or a metabolite thereof as a probiotic for intestinal bacterial population imbalance and intestinal regulation of metabolite concentration.
It is emphasized that while Lactobacillus plantarum DR7 has many of the medical functional uses described above, in other cases the strain needs to be used per medical use.
Another aspect of the invention relates to a composition comprising strain Lactobacillus plantarum DR7, wherein the strain is lyophilized in an amount of 10 in the composition 4 And 10 (V) 12 cfu/g; comprising metabolites of the strainWherein the metabolite comprises 2-hydroxyisocaproic acid and 3-phenyllactic acid.
The above mentioned medical uses for Lactobacillus plantarum DR7 and metabolites may optionally be formulated according to the use of the strain or its metabolites for the preparation of food supplements, medicaments, infant formulas, edible products or food products for the treatment and prevention of the above mentioned indications. Furthermore, methods for treating and preventing the above-mentioned indications may optionally be formulated comprising administering to a subject in need thereof an effective amount of a strain of the invention or a metabolite thereof. The subject of application is mainly mammals, mainly human bodies.
As used herein, the term "effective amount" refers to the number of colony forming units (cfu) of a strain in a composition, i.e., within the scope of sound medical judgment, this ratio is high enough to significantly improve the condition and thus treat it in a positive manner, but low enough to avoid serious side effects (at a reasonable benefit/risk ratio).
The terms used in the claims and aspects of the invention are to be understood in this description in their broad and common sense, and lactobacillus plantarum DR7 strain (Lactobacillus plantarum DR strain) is simply referred to as "DR7" in this description.
Description of the drawings
In order to facilitate an understanding of this patent, the drawings used in the embodiments herein are accompanied by corresponding illustrations to explain the associated drawings and to help better understand and understand the advantages embodied by the invention.
Figure 1 shows a graphical illustration of the occurrence and change of days of probiotic Lactobacillus plantarum strain DR7 (grey) or placebo (black) Upper Respiratory Tract Infection (URTI) administered over 12 weeks compared to week 0 in (a) young group (age <30 years; n=50), (b) adult group (age 30-59 years; n=52), and (c) all subjects (n=111), where p <0.05 and p <0.10.
Fig. 2 shows changes in cytokine levels (pg/mL; IL-1 beta, -4 and-10, tumor Necrosis Factor (TNF) -alpha, interferon (IFN) -gamma) in plasma over 12 weeks of probiotic Lactobacillus plantarum strain DR7 (grey) or placebo (black) administration in (a) young group (age <30 years; n=50), (b) adult group (age 30-59 years; n=52), (c) all subjects (n=111).
Fig. 3 shows graphs of iron ion reduction potential (FRAP) and thiobarbituric acid (TBA) levels in plasma, levels of Red Blood Cell (RBC) membranes and hemolysate (μm), in all subjects (n=111) in (a) and (d) young groups (age <30 years; n=59), (b) and (e) adult groups (age 30-59 years; n=52), (c) and (f) after 12 weeks of probiotic Lactobacillus plantarum strain DR7 (grey) or placebo (black) administration.
Fig. 4 shows the variation of T cell (CD 4, CD8, CD44, CD 117) and NK cell (CD 34, CD56, CD94, NKp46, NKp 30) related gene expression in plasma, administered Lactobacillus plantarum DR (grey) or placebo (black) over 12 weeks, in all subjects (n=111) in the (a) and (d) young groups (age <30 years; n=59), (b) and (e) adult groups (age 30-59 years; n=52), (c) and (f).
In figure 5.12 weeks, all subjects (total n= 109;Lactobacillus plantarum DR7,n =56; placebo group, n=53) were (a) pharyngeal; and (B) (nasal) symptoms and common influenza symptoms; plasma IFN-gamma (C), IL-10 (D), IL-4 (E) and thiobarbituric acid in plasma; a spearman correlation plot of changes in CD117, CD8 and CD44 gene expression in plasma.
Fig. 6 rats were treated for 12 weeks and behavioural assessment was performed using (a) the morris water maze and (B) the open field test (C) the T maze. The young group (normal diet), the young High Fat Diet (HFD) group (young rats high fat diet), the elderly group (D-galactose mediated aging; 600 mg/kg/day), the elderly High Fat Diet (HFD) group (D-galactose mediated aging high fat diet), the elderly high fat diet DR7 group (D-galactose mediated aging high fat diet plus L.plantarum DR7 log10 CFU/day), the elderly high fat diet statin (statin) group (D-galactose mediated aging high fat diet plus lovastatin) 2 mg/kg/day. The results are expressed as average values; error bars (SEM); n=6. The mean differences were significant for the same treatment group at different dates (P < 0.05).
FIG. 7.Lactobacillus plantarum DR7 (grey bars) or placebo (white bars) treatment effect for 12 weeks, numerical changes compared to week 0 (A) PSS-10, (B) stress, (C) anxiety, (D) depression, (E) total score DASS-42, in young groups (age <30 years), middle-aged (age >30 years) and all subjects. DASS-42 repeated measures ANOVA; pressure at age <30 years old; w: p=0.130; t: p=0.016, txw: p= 0.628; pressure of all subjects: w: p=0.003; t: p=0.036, txw: p=0.593; anxiety of age <30 years old: w: p=0.040; t: p=0.016, txw: p=0.291; anxiety with age >30 years old: w: p=0.127; t: p=0.045, txw: p=0.433; anxiety in all subjects: w: p=0.007; t: p=0.002, txw: p=0.170; total score for age <30 years: w: p=0.061; t: p=0.044, txw: p=0.484; total score for all subjects: w: p=0.005; t: p=0.028, txw: p=0.448. The P-value indicates that the different treatment groups are significantly different at each time point. Repeated measures ANOVA showed statistical differences on W: week number effect; t: DR7 and placebo effects; TXW: interactions between weeks and treatments. The results are expressed as average values; error bars (SEM); n=111 (dr7n=56, placebo n=55).
Fig. 8A and 8B. Cognitive and memory parameters were assessed by modified cog state simple cells, treated with placebo for Lactobacillus plantarum DR7 over 12 weeks, adult group (n=111; dr7n=56, placebo n=55).
FIG. 9 detection of hippocampal-related gene expression (A) gamma aminobutyric acid a subunit (GABA A2), (B) Tyrosine Hydroxylase (TH), (C) Dopamine Beta Hydroxylase (DBH), (D) Indoleamine Dioxygenase (IDO) and (E) tryptophan hydroxylase 1 (TPH 1) after 12 weeks of treatment. (F) Tryptophan is metabolized to kynurenine (kynurenine) via activation of IDO, or tryptophan is metabolized to 5-hydroxytryptamine (serotonin) via activation of TPH in the hippocampus. The young group (normal diet), the young High Fat Diet (HFD) group (high fat diet), the elderly group (D-galactose mediated aging; 600 mg/kg/day), the elderly high fat diet group (D-galactose mediated aging high fat diet), the elderly high fat diet DR7 group (D-galactose mediated aging high fat diet plus L.plantarum DR7 log10 CFU/day), and the elderly high fat diet statin group (D-galactose mediated aging high fat diet plus lovastatin 2 mg/kg/day). The results are expressed as average values; error bars (SEM); n=6.
Figure 10.Lactobacillus plantarum DR7 effect of relative expression of mRNA genes in blood after 12 weeks of treatment (normalized placebo as control): dopamine Beta Hydroxylase (DBH), tyrosine Hydroxylase (TH), tryptophan hydroxylase 1 (TPH 1), tryptophan hydroxylase 2 (TPH 2), 5-hydroxytryptamine receptor 6 (5-HT 6), indoleamine Dioxygenase (IDO), tryptophan 2, 3-dioxygenase (TDO), glutamate decarboxylase (GAD 65), gamma aminobutyric acid a-receptor a-5 (GABRA 5), brain Derived Neurotrophic Factor (BDNF) and cAMP response element binding protein (CREB). All subjects in (a) young group (age <30 years), (B) middle-aged group (age >30 years) and (C). P values showed significant differences between the different treatment groups at the respective time points. The results are expressed as average values; error bars (SEM); n=111 (dr7n=56, placebo n=55).
FIGS. 11A and 11B are photomicrographs showing (A) wild-type Drosophila (Oregon-R) hybridized to the vitropolymeric reporter (Glass Multiple Reporter) -GAL4 (GMR-GAL 4) to produce GMR-Orer eyes at 250X times and (B) normal shaped hexagonal eyes with straight bristles at 350X times. (C) Rough eyes were simultaneously porous, (D) malformed eyes lost hexagonal character, broken bristles from genetically modified drosophilse:Sup>A se:Sup>A x 042 (GMR-se:Sup>A x 142) at se:Sup>A multiple of 250 x 4, resulting from hybridization of GMR-se:Sup>A x 242 from the vitreous multimeric reporter-GA 4 (GMR-GA 4) with UAS-aβ42 expressing wild-type human gene se:Sup>A x 342, GMR-GA 4 causing degeneration of the whole retinse:Sup>A of the eye. Photomicrographs (E) Normal eye shape at 250 Xmagnification, (F) hexagonal eyes with slightly injured bristles at 350 Xmagnification from genetically modified Drosophilse:Sup>A Abetse:Sup>A 42 (GMR-Abetse:Sup>A 42) produced by hybridization of the vitropolymeric reporter-GA 4 (GMR-GA 4) with UAS-Abetse:Sup>A 42, wherein GMR-Abetse:Sup>A 42 is fed 100. Mu. 1X 10 11 CFU/mL Lactobacillus plantarum DR. (G) Quantitative statistics of drosophila eye morphology scores were calculated for each eye by the software flydynamics. High P values represent an increase in disorder status and a change in symmetry of the arrangement of the eyes, i.e. an increase in the phenotype of the eyes.
FIG. 12 rat menstrual flowHippocampal cytokine levels (A) interleukin 1-beta (IL-1B), (B) interferon gamma (IFN-gamma), (C) tumor necrosis factor a (TNF-alpha) after 12 weeks of intervention. Young age group (normal diet), young age group (high fat diet) elderly (D-galactose mediated aging; 600 mg/kg/day), elderly high fat group (D-galactose mediated aging high fat diet), elderly high fat DR7 group (D-galactose mediated aging high fat diet plus L.plantarum DR7 log) 10 CFU/day), senile high-fat statin group (D-galactose mediated aging high-fat diet plus lovastatin 2 mg/kg/day). The results are expressed as average values; error bars (SEM); n=6.
FIG. 13 relative expression levels of hippocampal apoptosis genes (A) tumor protein (P53), (B) tumor protein 19 (P19), (C) oversized B cell lymphoma (BCL-XL), (D) BCL2 in combination with X (BAX), (E) caspase 9 (CAS-9) after 12 weeks of treatment. (G) Apoptosis activators are produced in BAX, BCL-XL, CAS-9 interactions. The young group (normal diet), the young high-fat group (high-fat diet) elderly group (D-galactose-mediated aging; 600 mg/kg/day), the elderly high-fat group (D-galactose-mediated aging high-fat diet), the elderly high-fat DR7 group (D-galactose-mediated aging high-fat diet plus L.plantarum DR7 log10 CFU/day), and the elderly high-fat statin group (D-galactose-mediated aging high-fat diet plus lovastatin 2 mg/kg/day). The results are expressed as average values; error bars (SEM); n=6.
FIG. 14 cortisol (cotisol) and cytokines [ interferon gamma (IFN-gamma) in plasma; tumor necrosis factor alpha (TNF-a); interleukin (IL) -1 beta; IL-10; the level of IL-4 was compared to placebo (black gray) by the 12 week probiotic Lactobacillus p lantarum DR7 intervention group (a) young group (age <30 years), (B) middle aged group (age >30 years), (C) all subjects. Normalization of cytokines was detected by white blood cell count obtained from whole blood cell counts. The P-value was used to distinguish differences in treatment groups at different time points. The results are expressed as average values; error bars (SEM); n=111 (dr7n=56, placebo group n=55).
FIG. 15. (A) Panel shows phosphorylation activity of adenosine 5 'phosphate activated protein kinase (AMPK), lipid fraction in Dulbecco's modified minimal medium (DMEM) medium, blank MRS broth (de Mann, rogosa, sharpe (MRS) broth), lipid fraction in cell-free supernatant of strain Lactobacillus plantarum, protein fraction in blank MRS broth, protein fraction in cell-free supernatant of strain L.plantarium, polysaccharide fraction in blank MRS broth, and polysaccharide fraction in cell-free supernatant of strain L.plantarium. (B) Images illustrate the relative amounts of AMPK phosphorylation, commercial AMPK activator, 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), lactobacillus plantarum strain DR7 cell-free supernatant, lactobacillus plantarum strain DR7 cell-free supernatant, addition of commercial AMPK inhibitor, compound C, addition of compound C alone, and addition of lovastatin alone in a blank MRS broth medium.
FIG. 16 chromatogram (a) blank MRS broth (b) no Lactobacillus plantarum DR cell supernatant, high intensity peaks for 2-hydroxyisocaproic acid and 3-phenyllactic acid, expressed as HICA and PLA, respectively.
FIG. 17 blood of rats was collected after 12 weeks of treatment to measure telomere length (T/S ratio). Y (young controls), O (D-galactose mediated aging), O+S (D-galactose mediated aging atorvastatin 2 mg/kg/day), O+DR7 (D-galactose mediated aging plus L.plantarum DR7 (10 log CFU/day). Results are expressed as average; error bars (SEM); n=6.
Figure 18 treadmill fatigue test was used to test rats after a 12 week experimental period. Young (control), aged (D-galactose mediated Aged plus lovastatin 2 mg/kg/day), speed-DR 7 (D-galactose mediated Aged plus l.plantarum DR7 (10 log cfu/day) results were expressed as average; error bars (SEM); n=6).
FIG. 19A;19B and 19C. (A) assimilation of cholesterol by different lactic acid strains (1%, v/v inoculum size), MRS broth (containing 3% (w/v oxbile acid and 60. Mu.g/mL cholesterol)) was fermented for 24h at 37 ℃. Accumulation of cholesterol was co-cultured in (B) small intestine cells (HT-29) and (C) liver cells (HepG 2) for 24h at 37℃and cell-free supernatant fermentation broth (50%, v/v) of different lactic acid strains was added. Serum-free Darbeck Modified Eagle's Medium (DMEM) medium plus 1mM cholesterol With 0.03% oxgall acid. Blank MRS: sterile MRS broth medium was added to serum-free DMEM medium at 50% (v/v); 30242. intracellular cholesterol aggregation (D) and expression of beta-hydroxy-beta-mevalonate monoacyl-CoA (HMG-CoA) reductase (HMGCR) mRNA Gene (E) hepatocytes (HepG 2) were co-cultured with different concentrations of Lactobacillus plantarum DR-free cell supernatant for 24h at 37℃at 5% CO 2 . Non-treatment group: hepG2 was grown in serum-free Darbeck Modified Eagle Medium (DMEM) medium. Blank MRS: sterile MRS broth medium was added at 50% (v/v) to serum-free DMEM medium. (F) Beta-hydroxy-beta-mevalonate monoacyl-CoA reductase (HMGCR) mRNA gene expression (E) liver cells (HepG 2) and different concentrations of Lactobacillus plantarum DR-free bacterial supernatant were co-cultured for 3h at 37 ℃ at 5% CO 2 . AICAR (AMPK activator) and Compound C (Compound C) (AMPK inhibitor) served as positive and negative controls. Non-treatment group: hepG2 in serum-free Darbeck Modified Eagle Medium (DMEM) medium; blank MRS: hepG2 was added to serum-free DMEM medium at 30% (v/v) in sterile MRS broth medium; CFS-DR7, hepG2 was cultured in CFS from L.plantarum DR7 and added to serum-free DMEM medium at 30% (v/v). (G) The relative expression content of the liver cell (HepG 2) AMPK gene is cultured for 3h at 37 ℃ in different treatment groups, and 5% CO 2 . AICAR (AMPK activator) and compound C (AMPK inhibitor) served as positive and negative controls. Non-treatment group: hepG2 in serum-free Darbeck Modified Eagle Medium (DMEM) medium; blank MRS: hepG2 was added to serum-free DMEM medium at 30% (v/v) in sterile MRS broth medium; CFS-DR7, hepG2 was cultured in CFS from L.plantarum DR7 and added to serum-free DMEM medium at 30% (v/v). (H) Liver cells (HepG 2) of different treatment groups were phosphorylated with AMPK, cultured for 3h,37℃at 5% CO 2 . AICAR (AMPK activator) and compound C (AMPK inhibitor) served as positive and negative controls. Non-treatment group: hepG2 in serum-free Darbeck Modified Eagle Medium (DMEM) medium; blank MRS: hepG2 was added to serum-free DMEM medium at 30% (v/v) in sterile MRS broth medium; CFS-DR7, hepG2, was cultured in CFS from Lactobacillus plantarum DR, and added to serum-free DMEM medium at 30% (v/v). The results are expressed as average values; error bars (SEM); n is n=3. Statistical analysis was performed using one-way ANOVA, duncan's post hoc comparison. The average values are marked with different letters, with a significant difference (p<0.05). (I) Triglyceride (TG) levels in groups of 12 weeks of mediated aging rats treated with d-galactose (600 mg/kg/day). ND (normal diet), HFD (high fat diet), HFD-statin (high fat diet plus lovastatin 2 mg/kg/day), HFD-DR7 group (high fat diet plus L.plantarum DR7 log) 10 CFU/day), results are expressed as average; error bars (SEM); n=6.
FIG. 20 relative expression levels of the following genes (A) SCD1, (B) IL-6, (C) ABCG5, (D) ABCG8 in liver in groups of aged rats mediated by D-galactose (600 mg/kg/day) for 12 weeks. ND (normal diet), HFD (high fat diet), HFD-statin (high fat diet plus lovastatin 2 mg/kg/day), HFD-DR7 group (high fat diet plus L.plantarum DR7 log) 10 CFU/day), results are expressed as average; error bars (SEM); n=6.
FIG. 21 relative expression levels of genes below the liver (A) SR-B1, (B) LDL-R, (C) ABCA1, (D) ApoA1 in groups of rats treated with D-galactose (600 mg/kg/day) for 12 weeks. ND (normal diet), HFD (high fat diet), HFD-statin (high fat diet plus lovastatin 2 mg/kg/day), HFD-DR7 group (high fat diet plus L.plantarum DR7 log) 10 CFU/day), results are expressed as average; error bars (SEM); n=6.
FIG. 22 shows the relative expression levels of (A) AMPK.alpha.1 and (B) AMPK.alpha.2 below the liver in groups of rats subjected to D-galactose (600 mg/kg/day) for 12 weeks. ND (normal diet), HFD (high fat diet), HFD-statin (high fat diet plus lovastatin 2 mg/kg/day), HFD-DR7 group (high fat diet plus L.plantarum DR7 log) 10 CFU/day), results are expressed as average; error bars (SEM); n=6.
FIG. 23 liver anti-inflammatory cytokine IL-4 levels in groups of 12 weeks of D-galactose (600 mg/kg/day) treatment. ND (normal diet), HFD (high fat diet), HFD-statin (high fat diet plus lovastatin 2 mg/kg/day), HFD-DR7 group (high fat diet plus L.plantarum DR7 log) 10 CFU/day), results are expressed as average; error bars (SEM); n=6.
FIG. 24 at D-galactose (600 mg/kg/day)Liver HE stained sections in the group of 12 week-mediated aging rats. ND (normal diet), HFD (high fat diet), HFD-statin (high fat diet plus lovastatin 2 mg/kg/day), HFD-DR7 group (high fat diet plus L.plantarum DR7 log) 10 CFU/day), results are expressed as average; error bars (SEM); n=6.
FIG. 25 relative abundance of three dominant mycoplasmas. The method comprises the steps of carrying out a first treatment on the surface of the A) actinomycota, B) Bacteroides, C) hard wall fungus, D) ratio of hard wall fungus to Bacteroides in rat feces after 12 weeks of the test period. Young-ND: the young group normally eat; young-HFD: a high fat diet for young age groups; and (3) agent-ND: d-galactose mediated normal diet for the aging group; agent-HFD: d-galactose mediated aging group high fat diet; agent-HFD-statin: d-galactose mediated aging group high fat diet, 2 mg/kg/day of lovastatin; agent-HFD-DR 7: d-galactose mediated aging group high fat diet, L.plantarum DR7 log 10 CFU/day), results are expressed as average; error bars (SEM); n=6.
FIG. 26 analysis of the absolute content of acetic acid in feces. Young-ND: the young group normally eat; young-HFD: a high fat diet for young age groups; and (3) agent-ND: d-galactose mediated normal diet for the aging group; agent-HFD: d-galactose mediated aging group high fat diet; agent-HFD-statin: d-galactose mediated aging group high fat diet, 2 mg/kg/day of lovastatin; agent-HFD-DR 7: d-galactose mediated aging group high fat diet, L.plantarum DR7 log 10 CFU/day), results are expressed as average; error bars (SEM); n=6.
FIG. 27.12 analysis of the content of the first 70 water-soluble metabolites in rat feces after the test period; the map normalizes metabolites between relative values 2 to-2. Red represents higher concentration and blue represents lower concentration. Young-ND: the young group normally eat; young-HFD: a high fat diet for young age groups; and (3) agent-ND: d-galactose mediated normal diet for the aging group; agent-HFD: d-galactose mediated aging group high fat diet; agent-HFD-statin: d-galactose mediated aging group high fat diet, 2 mg/kg/day of lovastatin; agent-HFD-DR 7: d-galactose mediated aging group high fat diet, L.plantarum DR7 lo g 10 CFU/day).
FIG. 28.12 partial least squares analysis of water-soluble metabolites in rat feces after the test period (sPLS-DA). Young-ND: the young group normally eat; young-HFD: a high fat diet for young age groups; and (3) agent-ND: d-galactose mediated normal diet for the aging group; agent-HFD: d-galactose mediated aging group high fat diet; agent-HFD-statin: d-galactose mediated aging group high fat diet, 2 mg/kg/day of lovastatin; agent-HFD-DR 7: d-galactose mediated aging group high fat diet, L.plantarum DR7 log 10 CFU/day). D-galactose (600 mg/kg/day) was injected through the skin. Normal Diet (ND): standard ration, high Fat Diet (HFD): standard ration plus 25% fat.
Best mode for carrying out the invention
Lactobacillus plantarum DR 7A is isolated from fresh milk from the genus Dagne and obtained from Malaysia's private International (Malaysia) clinical nutrition. The strain is identified as Lactobacillus plantarum and is preserved in China general microbiological culture collection center (CGMCC) with an address of China national academy of sciences of China, national center for sciences of China, which is North Chenxi Lu No. 1, of the Korean area of Beijing, china. The collection number of the strain of the collection bacteria is CGMCC15535, and the collection time is 2018, 4 and 2 (02.04.2018). The deposit applicant was the clinical nutrition international (malaysia) private company limited and the prio. The deposited strain is active and has the desired conditions associated with its deposit.
Species identification was sequenced by 16S rRNA gene. 16S rRNA gene information and SEQ ID NO:1:
contig 25 (11475)
Contig length: 1475 base
Average length/sequence: 875 base
Total sequence length: 1751 base
Total strand (Total Str and): 1 sequence
Bottom strand (Bottom Str and): 1 sequence
Total: 2 sequence
Whole genome sequences were deposited under GenBank accession number CP031318 (2018, 9, 26) (modified historical accession number CP031318.1GI:1480141094 2018.9.26 07:35AM)
It is stated herein that by performing an experiment using the deposited strain as starting material, the skilled person can obtain variants or mutants of the strain according to general procedures, including conventional genetic mutagenesis and re-isolation techniques, while preserving the characteristics or enhancing characteristics of the original strain described in this patent. Thus, this patent discloses some of the possible mutants at the same time. As used herein, the term "variant" or "mutant" with respect to such strains refers to strains obtained from some naturally occurring or obligately evolving strain of the deposited strain, primarily to mutations, which still retain the function of the original strain. The technical approach to evaluating functions has been described in the "examples" section.
For example, a strain 'S "variant" 16S rRNA gene or whole genome sequence is expected to be 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the original strain' S16S rRNA gene SEQ ID NO 1 or CP031318 alignment.
In a particular embodiment, the mutant strain may be obtained by DNA recombination techniques. In another particular embodiment, the mutants may be obtained by random gene recombination. Thus, another aspect of the present patent relates to a method for obtaining a DR7 mutant strain, wherein gene mutation using the deposited original strain as a starting material, and obtaining the mutant strain and iterative change of biological functions based on the original strain will be described in detail.
In a detailed embodiment, the strain is fermented in an artificial medium and post-treated after fermentation to obtain a plurality of bacterial cells, which are then in a liquid medium or in a solid state. In detail, the post-treatment may be selected from the following modes: drying, freezing, freeze-drying, fluid drying, spray-drying and freezing of the liquid medium.
The strain is obtained by culturing or fermenting artificial culture medium under proper conditions. The term "artificial medium" is understood to mean a medium for microorganisms, which contains a natural matrix, and to which a chemical composition, such as polyvinyl alcohol, is added to regenerate serum function. The medium conventionally adapted is a nutrient broth medium comprising a basic carbon source (e.g. glucose), nitrogen source (amino acids and proteins), water and salts required for microbial growth. The growth medium is typically in liquid form or a solid medium prepared by adding agar and gel components. The strain can be cultured alone to prepare pure culture or mixed with other microorganisms, or mixed in a set proportion after being separately cultured. After cultivation, the strain may be used as pure bacteria, or bacterial cultures and cell suspensions may be used after suitable post-treatment, depending on the final purpose. Herein, "biomass" is understood to mean bacterial cells obtained after the microorganism has been cultured (or fermentation is synonymous with culture).
"post-treatment" has been described in the present invention wherein all procedures are directed to obtaining a target biomass of storable bacterial bodies. The post-treatment aims include reducing the metabolic activity of the somatic cells and delaying the proportion of apoptosis. The bacterial cells may be in solid or liquid form after post-treatment. In solid form, the somatic cells may be stored in a powdery or granular form. In either form, the cells in solid-liquid form are post-treated under artificial conditions and do not exist in their native form. Post-treatment is particularly characterized by the need for post-treatment reagents. The term "post-treatment agent" as used in this patent refers to a series of complexes that are used during post-treatment. The series of complexes includes, but is not limited to, dehydrating agents, bacteriostats, cryoprotectants (cryoprotectants), inert fillers (lyophilization supports), carrier materials (core materials), and the like, alone or in combination.
There are two basic methods to reduce the metabolic activity of the bacterial cells, and thus two sets of post-treatment modes are also formed. The first is to suppress the chemical reaction rate by lowering the temperature such as cold storage freezing, mechanical freezing, liquid nitrogen freezing. In addition, the biochemical reaction of the cells of the bacterium can be inhibited by a substrate inhibiting the growth of the bacterium, such as a bacteriostatic agent.
The second post-treatment method is to remove moisture from the biomass and sublimate the moisture by freeze-drying. The matching modes for removing the moisture of the biomass are drying, freeze drying, spray drying and fluidized bed drying. Post-treatment after drying, freeze drying, spray drying, fluid bed drying processes, the samples were finally processed to solid form.
The key in the post-treatment is freeze-drying, reducing the pressure to sublimate the water in the frozen cell suspension. The process includes three processes: the pre-freezing is carried out to form a freezing structure, most of water is removed firstly, and the second step of drying is carried out, and then bound water is removed. Because of variability in industrial culture and thallus collection, inert filling materials, i.e., lyoprotectants, are added for purposes and expectations. The function is to ensure the standard quantity of active probiotics in the product. The following list of useful commercial lyoprotectant materials: white granulated sugar, sucrose, lactose, trehalose, glucose, maltose, maltodextrin, corn starch, inulin, and the like. In addition, ascorbic acid, for example, is also commonly used as lyoprotectant. Whatever the material used, it is desirable to be able to ultimately be ground to the desired particle size, including powder formulation.
Medical application of Lactobacillus plantarum DR7
Lactobacillus plantarum DR7 has excellent properties as a probiotic. The probiotics need to have characteristics that are non-toxic, viable, adhesive and have a positive effect on health. Each strain is unique and cannot be analogized even with the strain to which it belongs.
In the experiments described in the examples section of this patent, the Lactobacillus plantarum DR strain was enumerated as having superior properties associated with improved health compared to the other.
1.Lactobacillus plantarum DR7 can be used to enhance the host's ability to resist upper respiratory tract infections by enhancing the immune system of the body, and this effect is demonstrated by a randomized, double-blind trial (see example 1 section, infra). The aim of this study was to explore Lactobacillus plantarum DR7 against upper respiratory tract infections (URT 1) to explore the relevant mechanisms under immune regulation. The DR7 strain (9 log cfu/d) was added for 12 weeks of randomized double-blind trial, and the volunteers participated in reached 109. Intake of DR7 reduced the symptoms and frequency of URT 1-induced rhinitis compared to placebo, observed after 12 weeks and 4 weeks, respectively. DR7 intake can promote expression of pro-inflammatory factors (IFN- γ, TNF- α) in plasma in the middle-aged group (30-60 years) while increasing the amount of anti-inflammatory factor (IL-4, IL-10) expression in the young group (< 30 years) while reducing pro-oxidative and oxidative stress levels in plasma compared to placebo. The young group showed higher expression levels of CD44 and CD117 in plasma after receiving DR7 compared to placebo, up to 4.50 and 2.22 fold, respectively. Meanwhile, the middle-aged group showed a decrease in the expression levels of CD4 and CD8 in plasma, 11.26 and 1.80 fold, respectively, indicating a decrease in the activation level of T cells. In contrast, both young and middle-aged groups of subjects were able to significantly increase the number of activated mature NK cells in plasma after receiving DR7 intake compared to placebo. Thus, DR7 alleviates the URT1 symptoms by just increasing the number of immune factors and enhancing immunity.
Thus, as previously mentioned, the Lactobacillus plantarum DR strain or metabolite component thereof referred to in this patent is useful for the prevention and treatment of upper respiratory infectious diseases by anti-inflammatory and immunomodulatory protection pathways.
2.Lactobacillus plantarum DR7 effect of stress and anxiety relief in the adult group also by random double blind, placebo controlled experiments demonstrated (see later example 2 for details) that 111 stress adults were recruited, as determined after screening by the adjusted PSS-10 questionnaire. DR7 (1×10) was ingested continuously for 12 weeks 9 cfu/day), reduced stress, anxiety symptoms were observed at week 8 compared to placebo, with a concomitant reduction in psychotic scores seen by DASS-42 panel. Compared to placebo, plasma intake of DR7 has reduced cortisol levels, pro-inflammatory factors such as interferon gamma, reduced transforming growth factor alpha, and plasma anti-inflammatory factors such as interleukin 10. DR7 increased adult @ compared to placebo and young subjects>Age 30) cognitive and memory functions such as primary attention, emotional cognition, and related learning abilities. Ingestion of D R7 can enhance the metabolism path of 5-hydroxytryptamine, reduce the contents of Dopamine Beta Hydroxylase (DBH), tyrosine (TH), indole 2, 3-dioxygenase and tryptophan 2, 3-dioxygenase in blood plasma, improve the contents of tryptophan hydroxylase-2 and 5-hydroxytryptamine receptor 6, and stably express TH and DBH through an experiment period of 12 weeks, so that the metabolism path of dopamine is stabilized. The result shows that DR7 can meet the requirements of FAO/WHO on probiotics as a natural strategy on physiological health of pressure adult masses in terms of improving physiological functions, cognition and memory.
Thus, as previously mentioned, as an aspect surrounding the patent content of Lactobacillus plantarum DR or its metabolites, it is useful for the prevention and treatment of stress, anxiety, memory and cognitive dysfunction, and the like.
3.Lactobacillus plantarum DR7 increases brain nerve health level, enhances neurotransmitter 5-hydroxytryptamine and dopamine metabolic pathways, and is able to combat Alzheimer's disease, hippocampal inflammation and apoptosis (see example 3 below for details). The behavioral evaluation study data in the old HFD rat group show that DR7 can reduce anxiety and enhance cognition. The concentrations of 3 pro-inflammatory factors in the hippocampus gradually increased in the aged mice, while a trend was found to be decreasing in the group of intake statin and DR 7. Both hippocampal neurotransmitter and apoptotic gene expression appear to reduce IDO and P53 expression, while increasing TPH1 expression in the older hyperlipidemic group that ingests DR7, suggesting a potential probiotic role for DR7 in the pathways associated with 5-hydroxytryptamine and oxidative stress aging. Thus, an effective embodiment is provided for Lactobacillus plantarum DR as a diet regulation strategy that can effectively enhance cognitive function during aging.
The 4.Lactobacillus plantarum DR7 strain exhibits a related pathological phenotype in the Drosophila melanogaster Alzheimer model that is effective in alleviating ocular neurodegeneration (see example 3, infra). DR7 strains simultaneously exhibit potential probiotic properties with AD reversal in the drosophila melanogaster alzheimer model. Under Lactobacillus plantarum DR intervention, the most prominent effect on drosophila was the ability to rescue the pathological phenotype of ocular surface roughness due to AD.
It follows that, as previously described, one of the important matters of this patent, surrounding Lactobacillus plantarum DR7 and its metabolites, is the prevention and treatment of neurodegenerative disorders. Specific examples are Alzheimer's disease, lactobacillus plantarum DR7 enhances neurotransmitter 5-hydroxytryptamine and dopamine metabolic pathways to counteract Alzheimer's disease as well as hippocampal inflammation and apoptosis.
5.Lactobacillus plantarum DR7 inhibits telomere shortening and enhances energy metabolism and thus combat aging (see example 5 below for details). Male SD rats were fed a high fat diet (54% kcal fat) while being injected with D-galactose diet for 12 weeks to make an aging animal model. The effect of the DR7 strain on the indexes of body injury caused by aging, such as telomere length, lipid peroxidation in blood plasma, liver AMPK expression, muscle performance and the like, is measured. Feeding statin and Lactobacillus plantarum DR (LP-DR 7) can significantly reduce the shortening of telomeres and increase the expression of AMPK-alpha 2 subunits. Under LP-DR7 intervention, AMPK-a 1 subunit amounts increased compared to the control group, whereas statin did not. In muscle performance tests, DR7 was found to increase running distance, duration, speed, work and ability compared to the control group, whereas statin did not.
It follows that, as previously described, one of the important matters of this patent around Lactobacillus plantarum DR7 and its metabolites is that energy metabolism can be shortened and enhanced by blocking telomeres. Is used for relieving physiological changes accompanied with aging. Manifestations of aging, such as reduced energy metabolism leading to impairment of locomotion and coordination, and impairment of the immune system due to oxidative reactions in the blood.
6.Lactobacillus plantarum DR7 reduces cholesterol by phosphorylation of AMPK and down regulates HMG-CoA reductase mRNA expression, thus regulating liver lipid and energy metabolism, effectively counteracting triglyceride elevation, inflammation and lipid accumulation (see example 6 below for details). Lactobacillus plantarum DR7 cell-free supernatant (CFS) also has the ability to metabolize cholesterol, reducing accumulation of HepG2 and HT-29 cell cholesterol, and simultaneously reducing expression of HepG2 cell HMG-CoA reductase (HMGCR) mRNA, as compared to other Lactobacillus species. The reduced expression level of HMGCR simultaneously attenuated the amount of AMPK inhibitor, and l.plantarum DR7 was hypothesized to perform its physiological role through the AMPK pathway in HepG2 cells, especially phosphorylation of AMPK instead of expression of AMPK mRNA. Summarizing the above, l.plantarum DR7 can perform its physiological function of cholesterol reduction through the AMPK pathway, in particular inhibiting HMGCR expression through phosphorylation of AMPK.
It follows that, as previously described, one of the important matters of this patent, around Lactobacillus plantarum DR7 and its metabolites, is the prevention and treatment of cardiovascular and cerebrovascular diseases and the reduction of cholesterol.
7.Lactobacillus plantarum DR7 altering lipid metabolism alleviating NAFLD in a model of aging rats (see example 6 below for details). In this study, different lactobacillus strains were used to evaluate whether hyperlipidemia and fatty liver can be alleviated by the AMPK pathway in senescent rats. Male SD rats were fed a High Fat Diet (HFD) and injected with D-galactose for 12 weeks to model aging. The treatment included i) a normal diet, ii) HFD, iii) HFD-statin (lovastatin 2 mg/kg/day), iv) HFD-Lactobacillus plantarum DR (10 log CFU/day). DR7 intake reduced triglyceride levels in serum over a 12 week experimental period compared to the HFD control group. The more prominent phenomenon observed in DR7 group was that both liver morphology and gene expression were significantly altered from the control group; down-regulating synthesis of lipid and beta-oxidation gene SCD1 in liver, up-regulating sterol excretion genes ABCG5 and ABCG8 in liver, further reducing sterol content in liver, and up-regulating energy metabolism genes AMPK alpha 1 and AMPK alpha 2 in liver. Summarizing the above, the study demonstrates that DR7 strain can enhance lipid disposition in the body by activating energy and lipid metabolism, suggesting that strain possesses the effect of relieving cardiovascular and cerebrovascular diseases and liver diseases by body metabolism.
It follows that, as previously described, one of the important matters of this patent around Lactobacillus plantarum DR7 and its metabolites is to prevent and treat hyperlipidemia and liver fat accumulation, and inflammation caused by lipid accumulation by hindering the rise of triglyceride levels. In particular, the strain can be used for preventing and treating nonalcoholic liver injury disease (NAFLD).
8.Lactobacillus plantarum DR7 has the effect of regulating intestinal flora balance and flora abundance and metabolite concentration (see example 7 below for details). Male SD rats were fed High Fat Diet (HFD) and injected with D-galactose for 12 weeks to model aging for evaluation of Lactobacillus plantarum DR7 effect on regulation of intestinal flora structure, short chain fatty acid and water soluble component content. As a result, DR7 was found to alter the diversity and structure of intestinal flora, as well as metabolites. Analysis of the water-soluble metabolites in feces revealed that L.plantarum DR7 intervention could result in increased metabolites such as tryptophan, leucine, tyrosine, cysteine, methionine, valine and lysine in feces.
It follows that, as described previously, one of the important matters of this patent around Lactobacillus plantarum DR7 and its metabolites is that as probiotics, the flora balance and flora metabolism can be regulated, and the intestinal microecological imbalance phenomenon can be alleviated.
Strains fulfill their probiotic functions involving a number of pathways and mechanisms, including the production of 2-hydroxyisocaproic acid (HICA) and 3-phenyllactic acid (PLA).
These metabolites can inhibit telomere shortening to slow down senescence signals. Further analysis, metabolites activate AMPK pathway by phosphorylating AMPK. Thus, the metabolites can directly regulate the body aging factors, and form a direct regulation relationship with the lipid metabolism related to the body aging factors. On the other hand, the anti-inflammatory and immunoregulatory effects on upper respiratory tract infection also promote the strain to play a role in slowing down and relieving aging. Therefore, the strain metabolite is suitable for people suffering from cardiovascular and cerebrovascular diseases, high cholesterol and the like caused by aging. It is worth mentioning that the regulation of the metabolites in terms of lipid metabolism is applicable to all mentioned conditions related to aging.
The unprocessed metabolite can be compounded with at least one component for further processing to form a healthy food or beverage. The processing needs to be gentle, allowing for maximum preservation of the nutritional components of the metabolites.
In another claim of the present patent, metabolites comprising HICA and PLA activate the phosphoadenosine-activated protein kinase (AMPK) pathway by phosphorylation, thereby slowing down physiological changes caused by aging. The metabolite is also a key metabolic enzyme upstream of e.g. 3-hydroxy-3-methylglutaryl coenzyme reductase (HMGCR). Under conditions of activation, AMPK phosphorylates a large number of downstream targets, resulting in e.g. up-regulation of the adenosine triphosphate pathway and down-regulation of the ATP utilization pathway. Furthermore, AMPK inhibits HMGCR gene expression, which is involved in cholesterol and isoprenoid production. All of the above indicate that ingestion of metabolites from the strain can lower cholesterol and thus both cholesterol and lipid levels in the host.
In addition, the remission action path of the aging phenotype is that macrophages are activated through AMPK phosphorylation, so that the infection of influenza viruses is reduced. Macrophages can be activated by cytokines such as gamma interferon, endotoxins such as lipopolysaccharides, and activated macrophages can destroy invading bacteria and infected cells. Activated macrophages are activated by releasing toxic compounds or proteins such as proteases, neutrophil chemokines, reactive oxygen species, cytokines, enoids and growth factors. Therefore, the metabolite in the patent can resist upper respiratory tract infection and has immunoregulation and protection effects.
Based on the above description, the metabolites disclosed herein may be used to enhance lipid metabolism. In particular, the metabolites upregulate ATP-binding cassette subfamily G member 5 (ABCG 5) and ATP-binding cassette subfamily G member 8 (ABCG 8) in the liver by downregulating stearoyl-coa dehydrogenase 1 (SCD 1), thereby lowering triglycerides in the serum. Down-regulated SCD1 inhibits the synthesis of single unsaturated fatty acids, also resulting in reduced triglyceride levels in plasma. It follows that the production of triglycerides in the body is limited by the activity of SCD 1. On the other hand, upregulated ABCG5 and ABCG8 maintain sterol balance by selectively driving neutral cholesterol secretion in bile. It follows that metabolites of the strain may reduce the occurrence of obesity-health risk conditions associated with aging factors.
Another specific disclosure referred to in this patent suggests that metabolites of complex HICA with PLA can slow the senescent phenotype through shortening of tissue telomeres. In a specific setting forth study, the high-fat diet group of complex metabolites was found to be capable of higher telomere single copy gene proportions than the high-fat diet group alone in the aging animal model. The active oxygen produced by mitochondrial oxidation is one of the important contributors to shortening telomeres. And the metabolites of the complex HICA and PLA may act on the mitochondrial oxidation process and reduce the production of reactive oxygen species during the process. In addition, the metabolites may reduce the β -amyloid polypeptide in brain tissue. Beta-amyloid polypeptides are key amino acids in dementia, progressive neurological dysfunction, synaptic function impairment, alzheimer's disease, and the like. Thus, the metabolites may hinder the formation of amyloid plaques in the brain.
Product form
The composition of the invention of the patent comprises 10 4 -10 12 cfu/g lyophilized form viable bacteria.
The composition of the invention also comprises the bacterial strain metabolites of 2-hydroxyisocaproic acid and 3-phenyllactic acid.
The number of viable cells effective in the application should be adjusted and determined by the practitioner according to the route and purpose of use, taking into account, for example, the age and physiological condition of the subject, the degree of the disorder, the final dosage form at the time of use, etc. When the oral route is adopted, the recommended daily dose of the strain composition of the invention is 10 7 -10 12 cfu, and meets the current regulatory requirements, the optimal oral dosage is 10 9 -10 11 cfu. "colony forming units" ("cfu") are defined as the number of clones of microorganisms present in an agar plate to calculate the number of bacterial cells. When the inventive composition of this patent is used, the ratio of strains is 1:1.
The application of the strain of this patent generally refers to the use of living cells, i.e., living cells, of the strain. Furthermore, it is also possible to expand to use the inactivated bacteria or bacterial lysate of the strain (obtained by changing pH, ultrasonic treatment, irradiation, high temperature and high pressure, etc.), and a composition comprising the beneficial factors produced by the strain of the present patent, for example, 2-hydroxyisocaproic acid and 3-phenyllactic acid in the metabolites.
The invention is particularly applicable to food supplements, medicaments, infant formulas, edible products and foods.
The application form of the invention can be that the composition is prepared into tablets, capsules, pills and the like for oral administration.
The composition of the invention can be prepared into any suitable form, and the biological activity of thalli and metabolites is not affected. Thus, when the form of use for preparing the inventive composition is designed for a particular end-of-use, it may be designed and prepared by a person skilled in the art of pharmacy and food technology.
The composition of the invention can also be prepared into a single component only containing thalli, and can also be mixed with one or more other active ingredients or pharmaceutical excipients, additives and food components. In particular applications of the inventive subject matter, one or more additional active ingredients may be added. When the other additive components are other types of probiotic bacterial strains, the application effect is better on the premise that the bacterial strain and the metabolite thereof do not generate antagonism. According to the above explanation, the cell of the present patent can be used in a pure bacterial form, can be used in a mixed form of cell culture, can be used as a partial component of cell culture, or can be used after post-treatment of cell culture, can be used together with a single strain or other strains, and can be used together with an auxiliary material by adding a suitable carrier. Other probiotics may also be used in combination.
The composition can be used in the form of pharmaceutical products. The definition of "pharmaceutical product" herein is relatively broad and includes any active ingredient added to the compositions of the present patent, and the cells of the bacterium are compatible with the matched agent. The "pharmaceutical product" is also not limited to medical agents. The term "pharmaceutical product" herein means a mixture, material, composition or the like, which is free from toxic harm to tissues of a human body or the like, irritation, and other side effects and complications such as allergic reactions, within sound medical judgment. Wherein each carrier or adjuvant, etc. must be compatible with the other compounds added to the final product. The matched carrier or auxiliary materials can be found in the standard data of pharmacy.
The pharmaceutical products have various forms and names according to different requirements of various countries. For example, a medicament may be a particular pharmaceutical product. Medical foods are also within the scope of the pharmaceutical products mentioned in this patent. Medical foods and special medical foods in some countries mean that certain foods can be effective for specific diseases through dietary intervention, and make up for the lack of nutrition in daily diets. This definition is defined by several laws, such as the amendment of the national administration of drugs of rare administration, issued by the U.S. food and drug administration 1988, european Commission directive 1999/21/EC. Medical foods are distinguished significantly from other broad categories of foods by the specific healthcare function they claim.
Probiotic compositions, such as the strains disclosed in this patent, are commonly used as food supplements. Food supplements are used as dietary supplements or nutritional supplements, often as a special pharmaceutical product. The food is prepared as a supplement to diet, and can provide nutrients lacking or insufficient in daily diet. Often, food supplements are also a food, but they are sometimes classified as pharmaceuticals, natural health products or nutraceuticals. For purposes of this patent, food supplements also include nutraceuticals. Food supplements are often sold over-the-counter as "over-the-counter medicines". If the food supplement is in the form of a pill or capsule, the same excipients as the drug are used. Food supplements are also in the form of foods that enhance certain nutritional utilities (e.g., infant formulas, etc.). Thus, for the specific application of the invention of this patent, it can be used as a food supplement.
The compositions of this patent may be mixed with suitable edible liquid or solid compositions and lyophilized to prepare tablets, pills, capsules, candies, granules, powders, suspensions, powders, syrups, and unit dosages thereof. Alternatively, the composition may be provided in a dose of lyophilized composition, and a liquid composition may be provided for administration by mixing and ingestion.
The compositions of the present patent may be added to a wide range of foods and edible products, such as infant milk. "edible product" is used broadly herein to include a wide variety of product types, including those described above, as may be utilized by animals; and products with acceptable sensory odors. A "food product" is understood to be an edible product that at the same time provides nutritional support to the body. Particularly, foods of interest today are food supplements and infant formulas. Suitable complexes in foods are those comprising, for example, oats, lactic acid fermented foods, resistant starches, dietary fibers, carbohydrates, proteins and glycosylated proteins. As a specific application in food, the thallus can be homogenized with other condiments, such as grains and milk powder to form infant formula.
One of the aspects of the present patent relates to a solid composition comprising a cryoprotectant; freeze-drying biomass of the strain; a carrier for compounding pharmacy. The pharmaceutical carrier may be selected from the group consisting of emulsions, gels, pastes, granules, powders, colloids. The present patent also provides oral health care products, in combination with pharmaceutical ingredients, edible products, dietary supplements and cosmetic products, which can be used in combination with the invention of the present patent at a certain effective dosage. The specific application product can be oral care product such as chewing gum, toothpaste, oral spray, cough relieving candy, and oral dispersible tablet. In particular, the pharmaceutical product component, the edible product or the dietary supplement may be applied in the form of a product of cough drops or orodispersible tablets.
Specific examples of compositions of the present patent invention are set forth below in example 8. Throughout the description and claims of "compositions" the diversity is not inclusive of other technical specifications, additives, ingredients, operating procedures. Other objects, advantages and features of the present patent will be apparent to those skilled in the art from a reading of the related specification, and can be learned by practicing the description of the present patent. Furthermore, the present invention includes all combinations of the specific and preferred embodiments described herein. The following examples and illustrations are illustrative of embodiments for a target application, but the present patent is not limited thereto.
MODE OF THE INVENTION
Examples
Materials and methods
Bacterial strains and cultures
Lactobacillus plantarum DR7 from fresh milk from Malaysia, gillene Hildebrand, obtained in a friendly manner from the company of the International (Malaysia) private Limited of clinical nutrition (Malaysia). The stored cell cultures were stored in a sealed condition at 20% glycerol (-20 ℃) and were passaged three times during activation using sterile MRS broth (Hi-Media, monbout, india) under 10% (v/v) inoculum size at 37℃for 24h per passage. The culture was centrifuged at 12000 Xg for 5min at 4℃and the pellet was resuspended in PBS (pH 7.5) to a final concentration of 1X 10 11 CFU/mL。
Description of the lipid component of DR7 metabolites
The removal of cell pellet at the bottom of the liquid medium is followed by the metabolite of DR7 in the resulting fermentation medium. The upper liquid medium is divided into lipid, protein and polysaccharide components. The lipid fraction was concentrated, hydrolyzed, and methyl esterified to Fatty Acid Methyl Ester (FAME) and analyzed by gas chromatography mass spectrometry (GC-MC). About 100. Mu.L of sample was mixed with 500. Mu.L of solvent consisting of 2% H in a 2mL centrifuge tube 2 SO 4 Is mixed with methanol for 2h at 80 ℃. Next, 500. Mu.L of a sodium chloride solution at a mass/volume ratio of 0.9% was added to the above sample tube together with 500. Mu.L of hexane, and centrifuged at 16000 Xg for 3min. The hexane layer was aspirated and injected into an Agilent 5977A gas chromatograph and 5977MSD mass spectrometry system combined device (Agilent; GC/MS) for the identification of FAME. A BPX-70 separation column was used.
In vitro experiments
Cholesterol assimilation
Methods for measuring the assimilation of cholesterol by lactobacillus have been previously described (Liong and Shah, 2005). The residual cholesterol in the supernatant was detected by a kit (Invitrogen, usa) according to the instructions.
Cell culture
HepG2 (human hepatoma cell line), and HT-29 (human colon cell line) were prepared using modified Dulbecco's modified Eagle's Medium (DMED) (Gibco, USA), previously described (Lew et al, 2013). HepG2 and HT29 cells were seeded in 12-well plates (1X 10) 5 Individual cells/well), DMEM-free serum plus 1mM cholesterol was incubated with 0.03% oxgall, 37 ℃,5% CO 2 24h. Cells were then eluted twice with phosphate buffered saline (PBS, pH 7.4). Lipid extraction was performed with 2mL of n-hexane to isopropanol (3:2) for 30min. The extract was collected in a glass tube and concentrated to dryness under nitrogen. The amount of cholesterol taken up and retained in the cells was measured by the kit and was performed according to the procedure described. Intracellular protein extraction the cells were placed in 12-well plates with 2mL NaOH (0.1N) and continuously shaken at 4 ℃ for 16h. Protein concentration was determined by Bradford method. Intracellular cholesterol accumulation is expressed in terms of the amount contained in a unit amount of intracellular protein.
Gene expression
Total RNA was extracted using TRISURE reagent (biological, UK) and reverse transcribed into cDNA, using reverse transcription kit (Thermo Scientific, USA) using random primers according to the kit instructions. Briefly, 1. Mu.g of total RNA was inverted and amplified in the reaction system at 2℃for 5min followed by 4℃for 60min. The reaction was terminated at 7℃for 5min. The cDNA was diluted 10-fold, used as a qPCR template with no nucleic acid water, and stored at-8 ℃. The mRNA levels of HMG-CoA reductase and AMPK were analyzed by real-time PCR, agilent AriaMx Realtime PCR System (Agilent Technologies, U.S.A.). The 20. Mu.l PCR reaction was performed using 10. Mu.l of 2X SensiFAST SYBR mix (biological, UK), 0.8. Mu.l of 10. Mu.M forward and reverse primer, and 1. Mu.l of cDNA. Primer sequences are shown in table 1, and amplification conditions were recommended by the manufacturer. The 18S rRNA gene was used as a housekeeping gene to analyze the data. mRNA expression levels were expressed as relative percentages of controls.
Table 1: primers in real-time PCR analysis
Cell-based ELISA for detection of AMPK Activity
AMPK activity was detected using a cell ELISA kit (RayBio cell-based protein phosphorylation ELISA kit, rayBiotech, usa) according to the instructions. Briefly, hepG2 cells (1×10 4 Individual cells/well) were seeded in 96-well plates, incubated at 3 ℃,5% co2, and no DMEM serum for 24 h. After that, the wells were rinsed twice with 200. Mu.L PBS, and 100mL fixative was added to each well and the wells were shaken at room temperature for 20min. Rinse 3 times with 200. Mu.L TBST, add 100. Mu.L quencher for 20min at room temperature, then add blocking solution for 1h at 3 ℃. mu.L TBST was washed 3 times, 50. Mu.L rabbit anti-phosphorylated AMPK-. Alpha.1/2 (Santa Cruz Biotechnology; thr172, 1:10000 dilution in a closed system) was added and incubated with shaking at room temperature for 3h. After elution, 50. Mu.L of HRP-labeled mouse anti-rabbit IgG (1:10000 dilution in a closed system; santa Cruz Biotechnology) was added and incubated for 1.5h at room temperature. Elution was performed 3 times, 50. Mu.L of TMB substrate (3, 5-tetramethylbenzidine) was added to each well and incubated for 30min with shaking in a dark room at room temperature. Finally, stop solution (H) 2 SO 4 2N), and the optical density value at 450nm was measured.
Animal experiment
Experimental group
All animal experiments were approved by the USM animal protection and use committee and (USM/animal ethical approval/2016/(724)). Male SD rats were divided into two groups of 8 weeks old based on two experimental animal models; 1) Aging model statin as positive control, or 2) aging and High Fat Diet (HFD) model statin as positive control.
In model (1), rats were divided into the following groups (n=6) for 12 weeks of experimental period: (1) young (control), (2) aging (D-galactose mediated aging), (3) aging-statin (D-galactose mediated aging, lovastatin 2 mg/kg/day), (4) aging-DR 7 (D-galactose mediated aging, L.plantarum DR7 (10 log CFU/day lyophilized powder with lyoprotectant 20% (v/v))).
In model (2), rats were divided into the following groups (n=6) for 12 weeks of experimental period: (1) The young group (normal diet), the young HFD (high fat diet of young rats), the elderly group (D-galactose mediated aging; 600 mg/kg/day), the aged HFD (D-galactose mediated aging high fat diet), the aged HFD DR7 group (D-galactose mediated aging high fat diet plus log 10 CFU/day), the aged HFD statin group (D-galactose mediated aging high fat diet plus lovastatin 2 mg/kg/day). Standard diet (Altromin, germany) was used for ND group, and 25% (w/w) animal fat (refined cream, 99% fat content) was added for high fat group. The therapeutic treatment was mixed into 1g of food feed and fed daily. Single cage feeding of rats ensures complete intake of the corresponding food. The experimental period was ended at 12 weeks, fasting was started 12 hours before the sacrifice, and the sacrifice was performed by the carbon dioxide method. All tissues were rapidly separated and washed with physiological saline. The tissues were used for real-time PCR gene quantitative analysis.
Telomere length detection
Two standard curves (telomeres and albumin) were generated by reference to DNA samples (standard DNA) and final DNA concentrations ranging from 0.073 ng/. Mu.L to 17.65 ng/. Mu.L. The T/S ratio of experimental DNA is T (ng of standard DNA sample versus experimental sample to calculate gene copy number of telomere template) divided by S (ng of standard DNA sample versus experimental sample to calculate gene copy number of albumin template). The T/S average was used to predict the average ratio of telomere length per cell.
Running machine fatigue test
Treadmill fatigue testing is referenced to Castro and Kuang (2017). The rats are firstly placed in a non-exercise room for 5min to adapt to the environment, and then run at a speed of 10m/min and 0 degree for 5 min. The rats were allowed to run on a 10 ° incline for 10min during a 2-3 day adaptation period. A grid with instantaneous microcurrent stimulation (0.4 mA) was placed behind the rats to stimulate the rats to run forward. On day 4 (test day), the belt speed was set at 10m/min,5min, and increased to 46m/min at a speed of 2 m/min. Rats continue running until either exhaustion (able to stand still against 5s of electrical stimulation) or until the highest speed of time is reached. Time, speed and distance are displayed and recorded when the rats are exhausted. The work and power calculations are based on the following equations: work (J) =body index (kg) ×gravity (9.81 m/s 2) ×vertical velocity (m/s×angle) ×time(s). Power (W) =work (J)/time(s).
Water maze test (MWM)
MWM were performed in a pool of 180cm non-reflective diameter and 51cm high. The pool was filled with water (19-20deg.C), divided into 4 zones (four-way regions of southwest and northwest), and a circular platform 20cm in diameter was set to feed 1.5cm of water under the north zone (D' Hooge and De Deyn, 2001). Each region was marked with a different subject and rat before the start of the evaluation. Prior to the formal test, rats were trained for 4 consecutive days per day for 4 rounds (each round was performed in four areas, southeast and northwest). Daily training was consistent. The rat is placed in a pool, faces the pool wall, and is launched into different areas each time, and the rat can swim freely within 60 seconds to find an escape platform. The time taken for the rat to find the platform was recorded.
Open Field Test (OFT)
OFT was modified in a previous study using a rectangular cassette (32 cm H. Times.38 cm W. Times.52 cm L) with a grid of labels (Sprott and Eleftherio, 1974). Each animal was gently transferred from the cage to the box and allowed to explore freely for 5min for 3 consecutive days. The number of times the rats entered the central and outer areas was recorded. The performance and trajectory of each rat was recorded by a camera (gobo, california, usa).
T maze experiment
Animals were subjected to a T maze experiment, which included a 3-arm maze with the main arm open and two closed arms 40cm high, 50cm long and 15cm wide. Each arm is held by a firm metal bracket 70cm from the ground. Animals were gently placed on the edge of the main arm as the starting point for the experiment. The experiment was divided into two phases, the first phase was to place food in the closed arms, allowing the animals time to walk freely into either closed arm for 60 s. After entry, the animals were blocked with cardboard for 30s, making them unable to leave the arm entered. The animals were then released and repositioned at the starting point for the second stage of testing. Animals were also given 60s time to access either closed arm, and the experiment was repeated 10 times daily, recording the number of times that arm was accessed.
Hippocampus tissue ELISA and gene detection
Rats were sacrificed and their hippocampal tissues were rapidly removed and rinsed with physiological saline. Total RNA isolation of tissue samples was placed in RNAlater (Sigma, misu, USA) until the samples were quick frozen and stored at-80℃for further ELISA and real-time PCR analysis.
Biochemical analysis
Whole blood was used to analyze serum lipid profiles (total cholesterol (TC), triglycerides (TG), low Density Lipoproteins (LDL) and High Density Lipoproteins (HDL)), liver functions (total protein, albumin, globulin, albumin/globulin ratio (AG ratio), aspartate Aminotransferase (AST), alanine Aminotransferase (ALT), alkaline phosphatase (ALP) and total bilirubin) and kidney functions (sodium, urea, chloride, potassium, creatinine, uric acid, calcium and phosphate). Tissues were further used for ELISA and real-time PCR gene expression analysis.
Liver histology
Samples were processed with fully automated tissue processing equipment (sammer technology Shandon Excelsior, usa). Samples were sectioned with paraffin-embedded cryomicrotomes, stained with hematoxylin eosin and observed under an optical microscope.
Analysis of metabolites in fecal samples
Water-soluble metabolite analysis was performed using gas phase mass spectrometry (GCMC) prior to sample diffraction according to Tsugawa et al 2011. Extraction of short chain fatty acids in feces is described in Nakajima et al 2017. The instrument platform was a GCMS-TQ8030 triple quadrupole mass spectrometer (Shimadzu, japan, kyoto).
Fecal flora sequencing and analysis
The V1-V2 region of the 16S rRNA gene in the extracted bacterial DNA was amplified. PCR products were prepared as in the previous study (Kato T et al 2014). The 16S rRNA gene sequencing was performed using the 454GS junction platform according to the manual. Sequencing results were analyzed with QIIME pipeline.
Clinical study of human body
Lactobacillus plantarum DR7 and placebo products
DR7 and placebo products were prepared by GN pharmaceutical private company (frank, malaysia). The product does not contain any components related to pigs or cattle; production is strictly GMP and has JAKIM (Islamic Malaysia)Development management). The probiotic product comprises 1×10 9 CFU/pouch DR7 and maltodextrin (95%), placebo only maltodextrin (100%). Each dose is 2g of pale yellow powder, aluminum paper is coated, and all products have the same taste.
The recruitment criteria were male and female ages 18-60 years, willing to adhere to the whole experimental period. Excluding diabetes mellitus of type one, with diseases requiring long-term administration, HIV/AIDS, glucose-6-phosphate dehydrogenase deficiency. All procedures involving human trials were passed by the ethical committee of the university of malaysia university clinical study group. Informed consent was also obtained for all subjects participating in the experiment.
With respect to brain studies, subjects evaluate their mental stress level with a modified Cohen sense stress test paper (PSS-10) (Cohen et al, 1983). Subject stress, anxiety, depression were evaluated using DASS-42 questionnaire (Lovibond and Lovibond, 1995), at baseline (week 0) and every 4 weeks ( weeks 4,8, 12). At the end of the experiment (12 weeks), the cognitive function of the subjects was assessed using a computer CogState simple Battery (Brief Battery) (CBB; mielke et al, 2015) analysis.
For the Upper Respiratory Tract Infection (URTI) study, subjects conducted two questionnaires: a basic condition questionnaire (baseline; 0 weeks), (ii) a health condition survey, recording the occurrence of upper respiratory tract infections (upper respiratory tract condition duration, number of symptoms, number of episodes) every four weeks. All questionnaires have three languages; english, malaysia, chinese (Lau et al, 2018a, b).
Human blood collection, gene and ELISA analysis
Blood sample collection time points were baseline period (week 0) and experimental end period (week 12), and the concentrations of the pressure hormones cortisol, IL-1β, -4 and-10, tumor Necrosis Factor (TNF) - α and Interferon (IFN) - γ were analyzed by ELISA. Genes involved in 5-hydroxytryptamine and dopamine secretion such as dopamine beta-hydroxylase (DBH), tyrosine Hydroxylase (TH), indole 2,3-dioxygenase and tryptophan 2,3-dioxygenase tryptophan hydroxylase-2 (trytophan 2,3-dioxygenasetryptophan hydroxylase-2) and 5-hydroxytryptamine receptor-6 were all analyzed by real-time PCR. Apoptosis and inflammatory gene expression in whole blood were both analyzed using the 18S rRNA gene as housekeeping gene.
Biochemical detection
A ratio analysis of plasma membrane to hemolysate of Red Blood Cell (RBC) samples was used to evaluate lipid oxidation and antioxidant properties. Lipid oxidation and antioxidant property analysis focused on analysis of Red Blood Cell (RBC) protoplasm, membranes, and hemolysates. RBC hemolysates and membranes were obtained as described in the previous study. Briefly, whole blood was centrifuged at 3500 Xg for 20min to separate plasma from erythrocytes. The red blood cells are washed by isotonic Tris-HCL buffer solution to remove the pale yellow shell layer, the isotonic Tris-HCL buffer solution is resuspended, and the centrifugation is carried out for 20min at 8000 Xg at 4 ℃ and repeated for several times until the cell membrane is faded. Finally, the faded pellet was collected and washed twice with Tris-HCL buffer, and finally heme-free RBC membranes (ghosts). The hemolysate is collected and concentrated during the procedure. RBC samples were prepared as 20 μl standards and were prepared using 20 μl undiluted plasma. Lipid peroxidation was detected by detecting Malondialdehyde (MDA), and antioxidant capacity was detected by the iron ion reduction/antioxidant capacity method.
Research on Drosophila Drosophila melanogaster Alzheimer's disease
The Drosophila used in this experiment was listed in the Drosophila database (http:// fybase. Bio. Indiana. Edu) provided by the Bloomington Drosophila collection (Blumeton, U.S.A): oregon-R wild type (# 5), vitropolymer reporter (Glass Multiple Reporter) -GA 4 (# 1104) (Moses and Rubin, 1991) and UAS-A.beta.42 (# 33769) (Sign and Mahoney, 2011). All the directed expression experiments used GMR-GAL4. For wild-type controls, oregon-R hybridizes to GMR-GA 4 to produce GMR-OreR, UAS-A.beta.42 hybridizes to GMR-GA 4 to produce the transgenic Drosophilse:Sup>A line GMR-A.beta.42, expressing A.beta.42. Stored at 25℃and hybridization temperature 29 ℃. Normal diet preparation, boiling 4% (w/v) corn starch, 5% (w/v) corn porridge, 10% (w/v) brown sugar, 0.7% (w/v) agar, 5% (w/v) heat sterilized yeast, 3% (w/v) methylparaben, and 0.7% (v/v) propionic acid were mixed, cooled and coagulated prior to delivery with sterile plastic bottles. All meals were free of methylparaben and propionic acid. Lactobacillus plantarum D R7 (100. Mu.L) at a concentration of 1X 10 11 The CFU/mL was added to the cooled and coagulated feed in an amount such that it was coagulated. Fresh DR7 feed was fed Drosophila within 2 hours of its setting.
5-10 Drosophila females (GAL 4 or UAS line) and 3-5 Drosophila males were placed in plastic bottles for mating prior to crossing. Wild control crosses were obtained from female Oregon-R lines crossed with UAS-A.beta.42 males, all GMR-A.beta.42 crosses were derived from UAS-A.beta.42 females crossed with GMR-GA 4 males. Control and transgenic GMR-aβ42.Nf Drosophila line normal diet feeding, no DR7 (table 2). The DR7 group used transgenic GMR-Aβ42Drosophila. The parent Drosophila is divided 4-5 days after the mating period. The F1 generation was obtained after a 10-day mating period and used for the next analytical experiment.
Table 2: drosophila series and experimental treatment group
| Drosophila line name | Probiotic bacterial strain | Short for short |
| OreR-GMR | Not eat | Wild type control |
| GMR-Aβ42 | Not eat | GMR-Aβ42.nf |
| GMR-Aβ42 | Lactobacillus DR7 | GMR-Aβ42.DR7 |
10 sub-generations were selected for each group and placed in McDowell-Trump fixative (Sigma-Aldrich) overnight at 4℃containing 4% formaldehyde 1% glutaraldehyde 0.1M phosphate buffer (Sigma) (pH 7.2). The specimens were washed three times with phosphate buffer and then fixed for 1 hour at 25℃using 1% (w/v) osmium tetroxide (sigma-aldrich). Washing the specimen with distilled water, and dehydrating the specimen by using gradient ethanol; 50%,75%,95% and 100% ethanol for 15min each. The dehydrated specimens were immersed in Hexamethyldisilazane (HMDS) (sigma) for 10min. The specimens were naturally air-dried overnight in a desiccator. The air-dried specimens were mounted, gold-plated, and then observed by a Scanning Electron Microscope (SEM) (SU 8010; hitachi Co., ltd., tokyo, japan). The eye area was calculated by the Flynogyper (http:// Flynogyper. Sourceforge. Net) method with each picture, and the phenotype score (P-value) calculated by picture J was based on hexagonally arranged eyes in the modified Drosophila eye (Yyer et al, 2018).
Results and conclusions
Example 1: lactobacillus plantarum DR7 effects of anti-inflammatory and immunomodulating protective effects against upper respiratory tract infections
Results of human clinical experiments
Human clinical trials were double-blind, randomized, placebo-controlled designed trials, with randomized trials also established on certain recruitment and exclusion criteria. Qualified subjects were randomly assigned by computer to two groups at 1:1, randomized list was done in a random block of 4, and age-stratified (18-29, 30-39,40-49,50-60 years), above which were ultimately assigned specific numbers to the probiotic group (DR 7) and placebo group (maltodextrin), respectively. The random assignment is performed by a statistically human, who is not in contact with the participating subjects. No member of the study group was aware of the study group represented by the assigned sequence number until the experiment was completed.
The calculation of the experimental sample size was based on authoritative considerations for the experimental design considering two parallel groups, one treatment group and one placebo group. A total of 124 subjects were required, 62 for both groups (DR 7 and placebo) and 10% of the additional recruits were withdrawn from the experiment. Such a calculation is based on the requirement that there is uncertainty in the continuous feedback of each individual control or experimental group subject, control versus subject 1:1, the probability (power) is 0.9, and the type I error probability associated with this zero hypothesis test is 0.05. Previous data indicate that probiotic intervention reduced the phenotype and duration of the pneumonia disease with a standard deviation of 1.7 days observed in the group and a reduction time of 1 day between the treatment group and the blank group.
The enrolled 124 subjects, 13 subjects withdrawn over 12 weeks, incomplete questionnaires, no blood samples were collected, so the final 111 volunteers completed the experiment for 12 weeks (56 in DR7 group, 55 in placebo group). The DR7 group and placebo group were significantly less different from the whole blood index in most phenotypes of their lower age groups such as the young (age <30 years), the adult group (age 30-59 years), as seen in table 3, but the hemoglobin concentration and basophils were higher in DR7 than in placebo group, while the eosinophils were higher in placebo group.
Table 3: baseline 111 (n=111) adult subjects were randomized to Lactobacillus plantarum DR or placebo groups for a 12 week double-blind trial.
*P<0.05
DR7 intervention slightly reduced (p < 0.10) the duration of the pathological phenomenon caused by influenza, such as nasal, pharyngeal etc. in the young group compared to placebo group (fig. 1A). DR7 intervention significantly reduced (p < 0.05) the duration of nasal symptoms in the adult group compared to placebo, slightly reduced the duration of common influenza symptoms and the frequency of upper respiratory tract infection occurrence (p <0.10; fig. 1B). DR7 significantly reduced the duration of nasal symptoms and the frequency of occurrence of upper respiratory tract infections (p < 0.05) over a 12-week experimental period compared to placebo while also having a relief effect on throat symptoms and general influenza symptoms (figure 1C) from all subjects.
The adult group showed a significant decrease in pro-inflammatory factors such as IL-1 beta, TNF-alpha, IFN-gamma in plasma (p < 0.05) compared to placebo during the 12-cycle trial with DR7 intervention, and DR7 reduced IFN-gamma in the young group (p <0.05; FIGS. 2A and B). The anti-inflammatory factor IL-4, IL-10 in plasma was significantly increased in the young compared to placebo group during the 12-cycle trial under DR7 intervention (p <0.05; FIGS. 2A and B), whereas the same phenomenon was found in the adult group. DR7 intervention reduced the pro-inflammatory factors TNF- α and IFN- γ in plasma compared to placebo, while increasing the levels of anti-inflammatory factors IL-4 and IL-10 in plasma in all subjects (p <0.05; fig. 2C).
While DR7 did not affect the antioxidant activity of plasma, RBC membranes and hemolysates in the young group compared to placebo (fig. 3A), DR7 slightly increased the antioxidant activity of RBC membranes in the adult group (p <0.10; fig. 3B), but it significantly increased the parameters as shown in the figures in the 12 week trial compared to placebo (p <0.05; fig. 3C). DR7 significantly reduced the concentration of Malondialdehyde (MDA) in the young, adult group, as judged by TBA (p <0.05; fig. 3d, e and F), over the 12 week experimental period, all subjects compared to placebo. DR7 intervention resulted in a slight decrease in TBA in RBC membranes over the 12 week experimental period compared to placebo group for all subjects (p <0.10; fig. 3D and F).
DR7 intervention upregulated CD44 and CD117 gene expression in young plasma over the 12 week experimental period compared to placebo group (p <0.05; fig. 4A). Meanwhile DR7 intervention down-regulated CD4 and CD8 gene expression in adult group plasma compared to placebo group over a 12 week experimental period (p <0.05; fig. 4B). For analysis at the overall subject level, no significant difference was observed between DR7 and placebo (fig. 4C). The young plasma levels of CD44 and CD117 were highly expressed in DR7, whereas the young plasma levels of CD4 and CD8 genes were low in DR7, and it was seen that activated T cells were less in DR7 than in placebo. DR7 intervention down-regulated NKp46 and NKp30 gene expression levels in young plasma, adult group CD56, NKp46 and NKp30, and CD56, CD94, NKp46 and NKp30 in all subjects (p <0.05; fig. 4d, e and F) compared to placebo group over a 12 week experimental period. It can be seen that DR7 intervention increased the number of non-dormant mature NK cells compared to placebo. These T cells and NK cell gene expression indicate that DR7 enhances mucosal epithelial cell integrity, reduces penetration of antigenic material, whereas NK cells in innate immunity are the first line of defense against foreign antigenic material and thus have not yet been required to activate large numbers of T cells in acquired immunity.
Figure 5 shows an analysis of the correlation of clinical symptoms of a subject with blood indicators.
Conclusion:
the purpose of this study was to investigate Lactobacillus plantarum DR7 against upper respiratory tract infections
(URTI) and related mechanisms of action in terms of its immunological properties. DR7 strain (9 log cfu/d) was used for a 12 week randomized, double-blind, placebo controlled group trial with 109 adults. DR7 intervention decreased the duration of nasal symptoms and the frequency of URTI occurrence after both 4 and 12 weeks compared to placebo. DR7 intervention promotes an increase in the number of pro-inflammatory factors (IFN- γ, TNF- α) in the plasma of middle aged population (30-60 years), increasing the number of anti-inflammatory factors (IL-4, IL-10) in young age group (age <30 years), while also reducing peroxidation and oxidative stress pressure levels in plasma compared to placebo group. The young group found 4.50 and 2.22 fold increases in plasma for CD44 and CD117, respectively, compared to the placebo group after receiving DR7 dry prognosis. Meanwhile, in the middle-aged group, the plasma CD4 and CD8 are reduced by 11.26-1.80 times compared with the placebo group after the DR7 is received, and the non-dormant mature NK cells are increased in the young group and the middle-aged group compared with the placebo group after the DR7 is received. Thus, DR7 alleviates symptoms of URTI by increasing the level of inflammatory factors and enhancing the effects of the immune response of the body.
Example 2: lactobacillus plantarum DR 7A is useful for the prevention and treatment of stress, anxiety, memory and cognitive dysfunction.
Results of animal experiments
The senile high-fat diet plus DR7 and statin groups showed that the delay time for finding the escape platform was shortened at 2 days before the experiment, whereas the blank senile high-fat group was found to have a shortened escape platform delay time (average difference-28.66; 95% CI-45.89 to-11.48; p < 0.05) 4 days before the experiment. The delay time for finding escape platforms under normal diet in young groups (average difference-8.75; 95% CI-26.48 to-8.93; p < 0.05) and in old groups (average difference-8.75; 95% CI-25.97 to-8.47; p < 0.05) appeared 3 days before the experiment (p < 0.05) FIG. 6A. The DR7 group rats have better memory and can take less time to find escape platforms.
The anxiety was evaluated using open field experiments, and the results showed that all groups did not have differences in the number of times they entered the outer ring region during the 3 day test period, but only the senile hyperlipidemia plus DR7 intervention group had reduced the number of times they entered the outer ring region 2 days prior to the experiment figure 6B, indicating a reduced anxiety level for this group.
The legend of fig. 6C shows that animals that were intervened by the l.plantarum DR7 strain improved the accuracy of entry into the T maze, indicating that the strain improved the animal's memory.
Results of human clinical experiments
In human clinical trial studies, DR7 intervention for 8 weeks reduced stress and anxiety levels compared to placebo, as assessed by DASS-42 questionnaire, see fig. 7. Thus, the strain is effective for reducing mental states such as anxiety and stress.
DR7 was intervened for 12 weeks compared to placebo, and memory and cognition was enhanced in the normal adult group as seen in figure 8.
Conclusion:
according to the modified PSS-10 questionnaire, 111 patients with stress were recruited. Intake of DR7 (1×10) 9 cfu/day) was 12 weeks, and at 8 weeks, a significant decrease in stress, anxiety, etc. mental index was found compared to placebo group as assessed by DASS-42 questionnaire. Plasma cortisol levels are also lower in the DR7 group than in the placebo group, with proinflammatory factors such as IFN-gamma and TGF-alpha being reduced and anti-inflammatory factor IL-10 being increased. With placebo group and young groupDR7 improved better than normal adult group (age>Age 30), such as basic attention, emotional cognition, associative learning, and the like. After the 12-week experimental period, intake of DR7 increased the 5-hydroxytryptamine pathway, reduced expression of dopamine β -hydroxylase (DBH), tyrosine Hydroxylase (TH), indoleamine-2, 3-dioxygenase and tryptophan 2, 3-dioxygenase in the plasma, increased expression of tryptophan hydroxylase-2 and 5-hydroxytryptamine receptor 6, enhanced dopamine metabolic pathway, and enhanced expression of TH and DBH compared to placebo. The result shows that DR7 meets the requirement of FAO/WHO on probiotics, and can be used as a natural way to promote the mental functions, cognition and memory health of people suffering from stress.
Example 3: lactobacillus plantarum DR7 the effects of hippocampal inflammation and apoptosis are resisted by enhancing the neurotransmitter 5-hydroxytryptamine and dopamine metabolic pathways.
Results of animal experiments
DR7 intervention did not significantly alter neurotransmitter pathways GABA (fig. 9A), dopamine (fig. 9B) or norepinephrine (fig. 9C). However, rats had reduced IDO expression in the hyperlipidemic group and increased TPH1 expression under DR7 intervention, and statins increased TPH1 expression in the hyperlipidemic group compared to the aging hyperlipidemic blank (mean difference-1.56; 95% CI-2.88 to-0.24; p <0.05; FIGS. 9E, F). This suggests that DR7 may act on the 5-hydroxytryptamine pathway to alleviate the anxiety symptoms of the hyperlipidemic group.
Results of human clinical experiments
In clinical trials, DR7 stem prognosis reduced stress and anxiety levels, namely dopamine β -hydroxylase (DBH) and tryptophan 2, 3-dioxygenase (TDO), both of which were important links to norepinephrine production, increased tryptophan hydroxylase-2 (TPH 2) and 5-hydroxytryptamine receptor 6 (5-HT 6) expression, both of which were involved in serotonin metabolic pathways and thus improved cognitive function, both of which were obtained in the young compared to placebo after 12-week experimental period (fig. 10A). A similar trend observed in the average adult group compared to the placebo group was that DR7 reduced DBH, tyrosine Hydroxylase (TH), tryptophan 2, 3-dioxygenase (TDO) expression with TDO, while increasing tryptophan hydroxylase-2 (TPH 2) expression (fig. 10B). For 12 weeks of experiment, DR7 intervention decreased expression of DBH, TH, TDO and TDO, and increased expression of TPH2 and 5-HT6 in all subjects (fig. 10C).
Fruit fly eye model research results
Drosophila's eye is an ideal research organization for neuro-developmental scientists, as it consists of simple neuroectoderms that constitute photoreceptors and appendages. Each eye consisted of 800 hexagonal cell eyes, forming a honeycomb similar to that formed by the arrangement of crystals. The eyes are arranged in rows and crossed into eyes to form a concave surface and an egg-shaped structure. Bristles with mechanical feel protrude at alternating apexes of each small eye pointing at a specific angle, providing a secondary sensing area. Heterologous expression of the human aβ42 gene in the compound eye, driven by the holoretinal GMR-GAL4, resulted in the generation of a neurodegenerative phenotype, i.e., a rough ocular phenotype (REP). Compared to the control OreR-GMR (FIGS. 11A, B), the GMR-Aβ42.Nf adult somatic eye severely distorts, i.e., the fusion of the ocular cells into voids causes the whole eye to assume a "glazed appearance" (FIGS. 11C, D). The number of hairs between the eyes is reduced and the pointing angle is random.
Feeding DR7 can reverse the eye pattern of GMR-aβ42 to varying degrees (fig. 11 EF) to conform to the control zone. The small eyes show the original hexagonal shape, and no fusion phenomenon and holes appear. In addition, the number of bristles is significantly increased and the angle is also adjusted to be the same. Morphological blemish quantification of Drosophila eyes, the phenotype score (P-value) of each eye was calculated by Flynogyper software. Higher P values represent increased disorder or altered symmetry of the small eye arrangement, and thus increased eye shape. Consistent with SEM pictures (fig. 11A-F) results, the P value of GMR-aβ42.Nf was significantly higher (p= 0.0355) than the wild-type control, indicating the negative effect of aβ42 on the Drosophila eye (fig. 11G). Feeding DR7 significantly reduced the P-value of the ocular form, indicating that DR7 can ameliorate drosophila neurodegenerative disease induced by GMR-aβ42.
Results of animal experiments
Intervention of the high lipid aging rat model with statin and DR7 was effective in reducing the concentration of 3 pro-inflammatory factors in the hippocampus is shown in FIG. 12 (average difference 5.62;95% CI 1.78 to 6.11; p < 0.05). The high level of TNF-alpha in the blood plasma of senile Alzheimer's disease patient plays an important role in the pathological changes of senile dementia, and the heightened IL-6 and TNF-alpha can be used as markers of cognitive decline, and IFN-g level is also increased in the aging process.
Results of human clinical experiments
In human clinical trials, DR7 reduced neuronal apoptosis in the hippocampus by regulating senescence and apoptotic gene expression (fig. 13). The BCL-XL, BAX and CAS9 genes govern the physiological processes of apoptosis and DNA damage (FIG. 13G). The senile hyperlipidemic group had higher BCL-XL, BAX and CAS9 gene expression than the senile control group, indicating that resistance and control of apoptosis induced by hyperlipidemic diet is required. The statin and DR7 intervention groups then underexpress these genes, indicating that DR7 and statin can inhibit the body's need for up-regulation of these genes, leaving the body under lower apoptosis and oxidative stress pressures.
After 12 weeks of experiment in the general adult population, DR7 was found to increase human anti-inflammatory cytokines while decreasing pro-inflammatory cytokines, see figure 14. The pressure frequently triggers human inflammatory response, DR7 reduces the pressure level of the young group human body and further reduces the related inflammatory index, and the pressure level and the inflammatory index of the common adult group population also have smaller reduction amplitude.
Conclusion:
the behaviours of rats in the high-fat aging group are studied by taking the rats as animal models, and the study proves that the intake of DR7 can reduce anxiety and enhance memory. The 3 pro-inflammatory cytokines in the hippocampus increased during aging, but fall back under statin intervention with DR 7. After the rat with high-fat aging group receives DR7 stem, the neurotransmitter and apoptosis genes in the hippocampus are expressed in a condition that IDO and P35 are expressed down, and the expression of TPH1 is increased, which indicates that DR7 has important regulation effect on 5-hydroxytryptamine and oxidative aging related pathways. It follows that these studies all provide examples for Lactobacillus plantarum DR7 as a future important dietary intervention strategy to promote cognitive function applications in the aging process.
The results of the Drosophila melanogaster AD model study indicate that DR7 possesses potential biological properties that reverse AD. The addition of probiotics can save the coarse eye pattern (REP) formed in the AD model, while the intervention experiments of Lactobacillus plantarum DR7 were found to have a more positive effect on the process.
Example 4: lactobacillus plantarum DR7 metabolite HICA and PLA and health efficacy thereof.
AMPK serves as an important biological switch governing many biological and metabolic pathways and thus also serves as an important targeting metabolite for assessing the biological function of DR 7. The effect of l.plantarum DR7 metabolism on 5' phosphoadenosine activated protein kinase (AMPK) phosphorylation was thus also studied. Dulbecco's modified minimal medium (DMEM) was used as a control for comparative analysis of the content ratio of lipid, protein and polysaccharide substances produced by L.plantarum metabolism. The fraction of lipid content isolated from l.plantarum metabolites is marked in fig. 15A to be seen as having higher AMPK phosphorylation activity, which is presumed to be closely related to lipid content. The lipid fraction was further purified and identified. Data comparing phosphorylation activity in native DR7 cell-free supernatant with commercial AMPK activator 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) is shown in fig. 15B. On the other hand, the phosphorylation activity of the l.plantarum metabolite was compared with commercial AMPK inhibitor, complex C and complex C added to cell-free supernatant. The ability of native DR7 cell-free supernatant to increase AMPK phosphorylation activity levels approximates AICAR, compromising its activity after complex C is added.
Lipid fraction detection in the l.plantarum DR7 metabolite was analyzed by GC-MS, different peaks compared to blank unfermented MRS medium (fig. 16). Two specific strong peaks are shown in chromatogram (b), 2-hydroxyisocaproic acid (HICA) and 3-phenyllactic acid (PLA), respectively. In terms of the anti-aging effect exhibited by DR7, HICA and PLA are important metabolites thereof for performing the anti-aging effect.
To verify that the two products were produced by DR7 metabolism, the metabolic pathways of the substance were evaluated. Leucine is a precursor substance that forms HICA, activated by HaDH enzyme based on the conversion of KICA. The gene transcribed HaDH was found in the DR7 complete gene sequence (protein ID AYA 79606.1). Phenylalanine is a precursor substance for PLA formation, activated by HPT, LDH, AAT enzymes based on PPA conversion. The gene for transcription of HPT (protein ID AYA 81108.1), the gene for transcription of AAT (protein ID AYA 79202.1), and the gene for transcription of LDH (protein ID AYA 79316.1) were found in the DR7 complete gene sequence. The discovery of these genes, HICA and PLA, both detected by FAME analysis, confirmed that HICA and PLA are metabolites of DR 7.
Example 5: lactobacillus plantarum DR7 an anti-aging effect by inhibiting telomere shortening and enhancing energy metabolism
After a period of 12 weeks, the telomere length was measured by collecting blood from rats, see fig. 17. The results showed that D-galactose mediated senescent rat telomere length was significantly lower than in young rats (P < 0.05). At the same time, the telomere length of the aged rats was also significantly lower than the aged macroarray (P < 0.05) with statin and DR7 intervention.
The treadmill fatigue test data were statistically analyzed to evaluate endurance of the rats after the 12-week experimental period. Running time and highest speed are calculated and estimated to obtain the maximum endurance value after exhaustion. As shown in fig. 18, the aging group endurance (distance, running duration, speed, work and power) was significantly lower than the young group (P < 0.05). The results showed that the work and power in the endurance assessment of the aging-DR 7 group was significantly higher than that of the aging group (P < 0.05). The aging-DR 7 group had significantly higher running distance and speed than the other group, and the running distance was significantly higher than in the aging group (P < 0.05).
Conclusion:
male SD rats were fed a high fat diet (54% kcal fat) while D-galactose was injected daily for 12 weeks to model aging. The effect of DR7 strain on defective lesions caused by aging was evaluated in terms of, for example, telomere length, lipid peroxidation in plasma, liver AMPK expression, and muscle performance. The intervention of statin and Lactobacillus plantarum DR (LP-DR 7) significantly reduces telomere shortening and promotes the expression of AMPK-alpha 2 subunit. AMPK- α1 subunit expression was also elevated with LP-DR7 intervention compared to the control group, whereas the statin group did not exhibit this function. Similarly, by comparing running distance, duration, speed, work and power metrics with control, it was found that DR7 intervention increased muscle performance, whereas statin group did not. In vivo experimental study of aging rat model shows that DR7 has effect of relieving defect pathological changes related to aging.
Example 6: lactobacillus plantarum DR7 effects of regulating liver lipid and energy metabolism on lowering triglyceride levels, inflammation and lipid accumulation
The screening of Lactobacillus strains was based on assimilation effort for cholesterol compared to the control (FIG. 19A; p < 0.05). The L.plantarum DR7 strain is one strain exhibiting high degradation to total cholesterol, and is subjected to subsequent analysis by using a colloid medium containing cholesterol. CFS of DR7 significantly reduced cholesterol accumulation in HT-29 cells (p < 0.05) compared to control (fig. 19b, c) while reducing cholesterol accumulation in HepG2 cells (p < 0.05) (about 19.7%) compared to control. CFS (CFS-DR 7) of the plantarum DR7 reduced cholesterol accumulation in HT-29 cells and HepG2 cells, followed by the next analysis. The liver is an important organ controlling cholesterol homeostasis, and CFS-DR7 at various concentrations was used to evaluate the accumulation of cholesterol and the expression of HMGCR mRNA (cholesterol synthesis rate-limiting enzyme) in HepG2 cells. Concentrations were set at 10% -50% (v/v), and concentrations above 50% also did not lead to apoptosis of the cells (data not shown). Lovastatin (1 μm) served as positive control. At all concentrations, cholesterol accumulation in HepG2 cells was significantly reduced, similar to the results for lovastatin (FIG. 19D; p < 0.05). However, only 30% concentration of CFS-DR7 promoted a decrease in HMGCR mRNA expression (FIG. 19E; p < 0.05). Thus, CFS-DR7 at 30% concentration was selected for further analysis. The effect of CFS-DR7 on the amount of HMGCR mRNA expression in the presence of AMPK inhibitor (Complex C, 10. Mu.M; as negative control), AMPK activator (AICAR, 1mM; as positive control) was investigated (FIG. 19F). CFS-DR7, 30% concentration, significantly reduced HMGCR mRNA expression (p < 0.05) compared to control, experimental conditions were 37 ℃ for 3h. This reduction in amplitude has a statistical advantage over the positive control (1 mM AICAR; p < 0.05), lovastatin (1. Mu.M) has no significant effect on the expression of HMGCR mRNA (p > 0.05). Complex C (10 μm) significantly increased HMGCR mRNA expression (p < 0.05) compared to control. CFS-DR7 treated HepG2 cells, in the presence of complex C (10. Mu.M), the expression of HMGCR mRNA was still significantly elevated compared to the control (p < 0.05), indicating a weak effect of CFS-DR 7. We further hypothesized that CFS-DR7 regulates HMGCR expression by enhancing AMPK expression. However, CFS-DR7 did not affect the expression of AMPK mRNA (FIG. 19G), indicating that this regulation was performed after transcription. Thus, we further analyzed the phosphorylation of AMPK by CFS-DR 7. In general, AMPK phosphorylation directs the activation of enzymes and dephosphorylation inactivates them. Our previous data show that CFS-DR7 significantly increases (p < 0.05) AMPK phosphorylation, comparable to the effect of AMPK activator (AICAR; fig. 18H), which was counteracted by the addition of AMPK inhibitor (compound C). CFS-DR7 significantly increased (p < 0.05) AMPK phosphorylation levels above lovastatin (1 μm). Here we propose that cholesterol metabolism can be altered by targeting the AMPK phosphorylation pathway using CFS derived from DR 7.
Such as DR7, shows a strong ability to regulate lipid metabolism in animal experiments in vitro. Serum Triglyceride (TG) levels values are shown in figure 19I after rats received a 12 week period of experiment. The results show a significant increase in triglyceride levels in rat serum (p < 0.05) upon ingestion of a High Fat Diet (HFD). Triglyceride levels were significantly reduced (p < 0.05) compared to the HFD alone group for HFD-DR 7. TG is an important marker in CVD and diabetes mellitus and non-alcoholic fatty liver disease (NAFLD).
Stearoyl-coa dehydrogenase 1 (SCD 1) is an enzyme that catalyzes the synthesis of a single unsaturated fatty acid (MUFA), a key control point in regulating liver lipid synthesis and beta oxidation. SCD1 enzyme was significantly (p < 0.05) down-regulated in the HFD-DR7 group (fig. 20A) compared to the HFD group. Thus, l.plantarum DR7 exerts its TG-lowering effect by down-regulating SCD1 gene expression. IL-6 gene expression was significantly reduced in HFD group (p < 0.05) compared to ND group, and IL-6 gene expression was significantly increased by L.plantarum DR7 intervention (p <0.05; FIG. 20B). ABCG5 and ABCG8 are half the size of ABC transporter and perform their function as dimers in regulating hepatic excretion of sterols (ABCG 5/G8). High-fat diet significantly reduced ABCG5 and G8 mRNA expression (p <0.05; fig. 20c, d), whereas l.plantarum DR7 intervention significantly upregulated ABCG5 and ABCG8 mRNA expression (p < 0.05).
HFD groups SR-B1 and LDL-R expressed significantly lower than ND groups (p <0.05; FIGS. 21A, B). The HFD-DR7 group significantly expresses SR-B1 and LDL-R (p < 0.05). ABCA1 mRNA expression was not significantly different between ND and HFD groups. However, the HFD-statin and HFD-DR7 groups significantly expressed ABCA1 (p < 0.05) more than the HFD group (FIG. 21C). Both statin and HFD-DR7 increased Apo-A1 levels compared to HFD (FIG. 21D).
mRNA of ampkα1 and ampkα2 was significantly reduced in both HFD groups compared to ND group (fig. 22). The HFD-DR7 group showed significantly higher expression of AMPKα1 and AMPKα2 than the HFD group. Thus, l.plantarum DR7 may activate AMPK by down-regulating SCD1 gene expression.
Figure 23 shows that IL-4 is significantly higher in the HFD group compared to the ND group. Compared to the HFD group; the IL-4 levels were lower in the HFD-statin and HFD-DR7 groups (p < 0.05).
There was a higher degree of lipid accumulation and fewer tissue structures in the HFD group compared to the ND group (FIGS. 24A, B). The HFD-statin and HFD-DR7 groups showed less lipid accumulation than the HFD group (FIGS. 24C, D). HFD-DR7 had better tissue architecture than the other experimental groups.
Conclusion:
lactobacillus plantarum DR7 cell-free suspension (CFS) shows high ability to assimilate cholesterol, reduces accumulation of cholesterol in HepG2 cells and HT-29 cells, and reduces expression of HMG-CoA reductase (HMGCR) mRNA by HepG2 cells. The HMGCR expression reduction phenomenon is offset by the occurrence of AMPK inhibitors, presumably l.plantarum DR7 performs its function through the AMPK pathway, especially by enhancing AMPK phosphorylation in HepG2 cells rather than by affecting AMPK mRNA expression. Summarizing the above, the study demonstrates that l.plantarum DR7 exerts its cholesterol lowering effect through the AMPK pathway, in particular through AMPK phosphorylation leading to reduced HMGCR expression.
DR7 was used to verify whether it acts via the AMPK pathway against a model of aging rats that alleviate hyperlipidemia and fatty liver disorders. Male SD rats were subjected to a High Fat Diet (HFD) for 12 weeks with daily injections of D-galactose to construct an aging animal model. Experimental treatments included i) a normal diet, ii) HFD, iii) HFD-statin (lovastatin 2 mg/kg/day), iv) HFD-Lactobacillus plantarum DR (10 log CFU/day). Over a 12 week experimental period, l.plantarum DR7 intervention reduced serum triglyceride levels compared to the HFD control group. The more positive effect of l.plantarum DR7 intervention was found from gene expression in liver tissue compared to the control; down-regulating liver lipid synthesis and beta-oxidation gene SCD1, up-regulating liver sterol secretion genes ABCG5 and ABCG8, lower liver fat, up-regulating liver energy metabolism genes AMPK alpha 1 and AMPK alpha 2. Summarizing the above, the study demonstrates that DR7 strain enhances energy and lipid metabolism activity, and improves lipid metabolism, and shows that DR7 has good application prospect as a natural intervention approach for relieving cardiovascular and liver diseases.
Example 7.Lactobacillus plantarum DR7 effects on intestinal flora imbalance and regulation of metabolite concentration.
During aging, the number of actinomycetes gradually decreased, whether HFD was present or not (p <0.05; FIG. 25 a). DR7 intervention prevented the decrease in the number of actinomycetes in aged rats fed HFD (p < 0.05). Actinomycetes, and its primary genus bifidobacteria, are generally considered to be related to healthy intestinal flora configurations. Thus, DR7 promotes health in the gut of aging rats. Bacteroides were higher in the aging-ND group (p < 0.05) compared to the young-ND group, whereas teichoic acid was not significantly different in all treatment groups (fig. 25b, c). D-galactose induced generation of the ratio of hard wall to Bacteroides (F/B ratio) was significantly reduced (p < 0.05), whereas L.plantarum DR7 intervention significantly increased the F/B ratio in HFD senescent rats and was similar to the young-ND group (FIG. 25D).
Acetate concentration in the faeces of high fat diet rats was significantly reduced. Statin and l.plantarum DR7 group exhibited higher acetate concentrations (fig. 26). Acetate is often reported to have reduced levels of fatty acids, cholesterol, and insulin sensitivity in plasma as well as liver. Acetate has also been reported to lower the pH in the lumen, inhibit pathogenic proliferation, and maintain intestinal barrier function.
Metabolites of the intestinal flora have a direct effect on host health and disease. L. plant DR7 intervention can increase the levels of essential amino acids such as tryptophan, leucine, tyrosine, cysteine, methionine, valine and lysine in feces (figure 27), which are critical for amino acid balance and health in the host. As shown in fig. 28, l.plantarum DR7 intervention can alter the distribution of intestinal flora.
Conclusion:
male SD rats were continuously fed a high-fat diet for 12 weeks while D-galactose was injected to construct an aging model. Lactobacillus plantarum DR7 the effect of short chain fatty acids on fecal flora structure was studied with water soluble components. As a result, it was found that strain intervention can alter the diversity and composition of the intestinal flora, either at the phylum level or at the metabolic level. Analysis of the water-soluble metabolites in the feces revealed that L.plantarum DR7 intervention could enhance the metabolism of amino acids such as tryptophan, leucine, tyrosine, cysteine, methionine, valine and lysine.
EXAMPLE 8 composition containing Lactobacillus plantarum DR7
TABLE 4 Table 4
Remarks: the materials and methods used in this section in the context of the articles mentioned above are incorporated by reference in their entirety.
Free text of sequence Listing
Sequence listing
Claims (16)
1. Lactobacillus plantarum strainLactobacillus plantarum) The strain is preserved in China general microbiological culture collection center (CGMCC), the preservation number is CGMCC15535, the whole genome sequence is uploaded in GenBank, the accession number is CP031318, and the metabolite comprises 2-hydroxy isocaproic acid and 3-phenyllactic acid.
2. The strain according to claim 1, wherein the strain or a metabolite thereof is used for the prevention or treatment of upper respiratory tract infections.
3. The strain according to claim 1, wherein the strain or a metabolite thereof is used for preventing and treating memory and cognitive dysfunction, stress and anxiety.
4. The strain according to claim 1, wherein the strain or a metabolite thereof is used for preventing and treating neurodegenerative diseases.
5. The strain according to claim 4, wherein the neurodegenerative disease is Alzheimer's disease.
6. The strain, strain or metabolite thereof according to claim 1 for use in slowing the signs of aging.
7. The strain according to claim 1, wherein the strain or a metabolite thereof is used for the prevention and treatment of hyperlipidemia, liver fat accumulation and fat metabolism.
8. The strain according to claim 7, wherein the strain or a metabolite thereof is used for the prevention and treatment of nonalcoholic fatty liver disease.
9. The strain according to claim 1, wherein the strain or a metabolite thereof is used for preventing and treating cardiovascular diseases and reducing cholesterol.
10. The strain according to claim 1, for use as a probiotic for intestinal regulation of intestinal flora imbalance and metabolite concentration.
11. A composition comprising the strain of claim 1, wherein the strain is freeze-dried and the amount in the composition is 10 4 And 10 (V) 12 cfu/g.
12. The composition of claim 11, comprising a strain metabolite, wherein the metabolite comprises 2-hydroxyisocaproic acid and 3-phenyllactic acid.
13. The composition according to any one of claims 11-12, in a form selected from the group consisting of: food supplements, pharmaceutical agents and infant formulas.
14. A composition according to any one of claims 11-12 in the form of an edible product.
15. The composition according to any one of claims 11-12, in the form of a food product.
16. The composition of claim 13 in the form of a tablet, capsule or pill.
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| PCT/KR2019/011130 WO2020050553A1 (en) | 2018-09-03 | 2019-08-30 | Probiotic strain for upper respiratory tract infection, stress, anxiety, memory and cognitive dysfunctions, and ageing |
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| WO2022094178A1 (en) * | 2020-10-30 | 2022-05-05 | The Regents Of The University Of California | Compositions and methods for diagnosing and treating patients with a history of early life adversity |
| US20240058398A1 (en) * | 2020-12-16 | 2024-02-22 | Meiji Co., Ltd. | Composition for improving inflammation of brain tissue |
| CN112915109B (en) * | 2021-01-21 | 2023-03-24 | 吉林省农业科学院 | Application of lactobacillus plantarum DP189 in preparation of medicine for preventing and/or treating Alzheimer disease |
| CN113337428B (en) * | 2021-06-03 | 2023-06-06 | 海南大学 | Lactobacillus plantarum HNU082 and application thereof |
| CN113337427A (en) * | 2021-06-03 | 2021-09-03 | 海南大学 | Lactobacillus plantarum HNU082, composition and application thereof |
| WO2023230513A1 (en) * | 2022-05-26 | 2023-11-30 | Pouda Biotech Llc | Lactobacillus plantarum pda6 having neurological effects and compositions including the same |
| CN115181695B (en) * | 2022-06-27 | 2023-08-29 | 广东粤港澳大湾区国家纳米科技创新研究院 | Lactobacillus plantarum5b4m2 and application thereof |
| CN115838653A (en) * | 2022-07-11 | 2023-03-24 | 四川维创天益生物科技有限公司 | Lactobacillus plantarum GM11 for improving bad mood and application thereof |
| CN115089619A (en) * | 2022-07-18 | 2022-09-23 | 南昌大学 | Application of lactobacillus plantarum ZDY2013 in preparation with function of relieving non-alcoholic fatty liver disease |
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| CN105505814B (en) * | 2015-11-30 | 2020-01-17 | 南昌大学 | Lactobacillus plantarum for delaying senescence |
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Non-Patent Citations (5)
| Title |
|---|
| Identification of 2-hydroxyisocaproic acid production in Iactic acid bacteria and evaluation of microbial dynamics during kimchi ripening;PARK B，等;《Scientific reports》;20170907;第7卷;第1-8页 * |
| Lactobacillus plantarum DR7 for gut-brain-axis benefits(DR7);Clinicaltrials.gov;《https://clinicaltrials.gov/ct2/show/NCT03370458》;20180703;第1-7页 * |
| Lactobacillus plantarum DR7 Reduces Cholesterol via Phosphorylation of AMPK That Down-regulated the mRNA Expression of HMG-CoA Reductase;Lew Lee-Ching，等;《Korean journal for food science of animal resources》;20180430;第38卷(第2期);第350-361页 * |
| Production and characterization of an antifungal compound (3-phenyllactic acid) produced by Lactobacillus plantarum strain;PREMA P，等;《Food and Bioprocess Technology》;20101231;第3卷;第379-386页 * |
| Role of Lactobacillus plantarum MTCC1325 in membrane-bound transport ATPases system in Alzheimer’s disease-induced rat brain;MALLIKARJUNA N，等;《BioImpacts》;20161231;第6卷(第4期);第203-209页 * |
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