Intestinal flora regulator and application thereof
阅读说明：本技术 一种肠道菌群调节剂及应用 (Intestinal flora regulator and application thereof ) 是由 张全斌 邓真真 王晶 吴宁 岳洋 耿丽华 于 2021-09-30 设计创作，主要内容包括：本发明公开了一种褐藻褐藻来源的肠道菌群调节剂及应用,该调节剂主要为岩藻多糖,能够调节肠道菌群的结构组成,增加益生菌的相对丰度,维持肠道屏障的完整性并改善肠道细菌的代谢。该肠道菌群调节剂在动物实验水平上已经验证能显著的增加肠道中微疣菌门(Verrucomicrobia)的比例,增加肠道益生菌Akkermansiaceae和Akkermansia-muciniphila的相对丰度,维持肠粘膜的厚度,增加短链脂肪酸(SCFAs)的产生。该肠道菌群调节剂够降低高脂饮食诱导的代谢紊乱小鼠的高血糖、胰岛素抵抗、高脂血症状；保护肝肾功能,减少高尿酸症及非酒精性脂肪肝的发生；并降低机体炎症反应。该肠道菌群调节剂在制备治疗或预防代谢紊乱性疾病的药品、食品和/或保健品中有广泛的应用。(The invention discloses an intestinal flora regulator derived from brown algae and application thereof. The intestinal flora regulator has been verified on an animal experimental level to remarkably increase the proportion of Verrucomicrobia (Verrucomicrobia) in intestinal tracts, increase the relative abundance of intestinal probiotics Akkermansia and Akkermansia _ muciniphila, maintain the thickness of intestinal mucosa and increase the generation of short-chain fatty acids (SCFAs). The intestinal flora regulator can reduce hyperglycemia, insulin resistance and hyperlipidemia symptoms of mice with metabolic disorder induced by high fat diet; protecting liver and kidney function, and reducing hyperuricemia and non-alcoholic fatty liver; and reducing inflammatory response in the body. The intestinal flora regulator can be widely applied to preparation of medicines, foods and/or health products for treating or preventing metabolic disorder diseases.)
1. An intestinal flora modulator, characterized in that: the main component of the product is low molecular weight fucoidin.
2. The modulator of claim 1, wherein: the intestinal flora regulator is prepared from one or more of marine brown algae including herba Zosterae Marinae, thallus laminariae, Sargassum, sea oak, cornu Cervi, Ascophyllum nodosum, thallus laminariae, Cladosiphon okamuranus, Sargassum thunbergii, Sargassum integerrimum, Cyrtymenia Sparsa, Sargassum Ammoniacus, semen Panici Miliacei, and Sargassum pallidum.
3. The modulator of claim 1, wherein: the intestinal flora regulator contains fucose 30-40%, sulfate 25-35%, and molecular weight of 3-9 kDa.
4. Use of a modulator according to claims 1-3 for improving the structure of the intestinal flora, characterized in that: the application of improving the structure of the intestinal flora, namely increasing the relative abundance of specific probiotics Akkermansia _ muciniphila, maintaining the thickness of intestinal mucosa and increasing secondary metabolites of the intestinal flora.
5. Use according to claim 4, characterized in that: the structure for improving the intestinal flora is to increase the proportion of Verrucomicrobia (Verrucomicrobia) in the intestinal tract and increase the ratio of bacteroidetes to firmicutes.
6. Use according to claim 4, characterized in that: the intestinal probiotics are Akkermanspiaceae and Akkermansia _ muciniphila.
7. Use according to claim 4, characterized in that: the secondary metabolite of the intestinal flora is short-chain fatty acid.
8. Use of the intestinal flora modulator according to claims 1-3 in food, pharmaceutical, health care products for the treatment and prevention of symptoms/diseases associated with metabolic disorders.
9. The symptoms/diseases associated with metabolic disorders according to claim 8 include obesity, hyperglycemia, insulin resistance, hyperlipidemia, diabetic nephropathy, non-alcoholic fatty liver disease, inflammatory bowel disease, metabolic syndrome and the like.
10. Use according to claim 8, characterized in that: the food, medicine, health product and biological material for treating and preventing metabolic disorder related symptoms/diseases can be added with pharmaceutically and biologically acceptable carriers or auxiliary materials.
Technical Field
The invention belongs to the field of food health care, and particularly relates to an intestinal flora regulator derived from brown algae and an application thereof.
Background
The key role of the intestinal flora in maintaining health has attracted considerable interest. The human microbiota is mainly composed of four phyla: actinomycetes (Actinobacillia), Firmicutes (Firmicutes), Proteobacteria (Proteobacteria) and Bacteroides (Bacteroides). It is estimated that there are up to 1000 different bacteria in the intestine, including nearly 200 million genes. In fact, the number of bacteria in the gastrointestinal tract is about 10 times the total cell number of the human body, which makes the total genome of bacteria much larger than the human genes. Thus, humans are also referred to as "super organisms". Based on the symbiotic relationship between intestinal bacteria and human, the intestinal flora is closely related to the physiological state of human body. Among them, the intestinal flora plays a crucial role in metabolic diseases, and may become a potential drug target for preventing and treating the diseases. The intestinal flora is an important environmental factor for regulating weight and energy homeostasis, and plays an important role in aspects of blood glucose homeostasis, impaired fasting blood glucose, type 2 diabetes, insulin resistance and the like. This "external" organ regulates the intake and storage of energy in the body through various metabolic functions and different control regimes.
Algal polysaccharides have been proposed as supplements for health enhancement and disease management. In recent years, fucoidan has become a potential adjuvant for the treatment of metabolic syndrome due to its broad activities of anti-oxidation, immunomodulation, anti-inflammation and anti-obesity. In addition, as a dietary fiber of marine origin, the regulation effect of fucoidan on intestinal flora is also gradually attracting attention. Fucoidan has been shown to alleviate metabolic disorders including lowering blood glucose, lowering blood lipid, and resisting obesity by altering the intestinal microbiota. It is contemplated that there may be structural dependence of fucoidan on the regulation of gut flora. Fucoidan with different structures also has different effects on intestinal flora. The invention discloses application of fucoidin serving as an intestinal tract regulator for specifically increasing probiotics Akkermansia _ muciniphila in improving the structure of intestinal tract flora, improving the relative abundance of intestinal tract probiotics, maintaining the thickness of intestinal mucosa and increasing secondary metabolites of the intestinal tract flora and application of the fucoidin in foods, medicines and health-care products for treating and preventing symptoms/diseases related to metabolic disorder.
Disclosure of Invention
The invention aims to provide an intestinal tract flora regulator derived from brown algae and application thereof. Animal experiments show that the intestinal flora regulator can improve intestinal flora disorder and metabolic disorder induced by high fat diet.
Further, the content of fucoidin in the intestinal flora regulator is 30% -40%, the content of sulfate group is 25% -35%, and the molecular weight range is 3kDa-9kDa, wherein the optimal value is 7 kDa. The intestinal tract regulator is prepared from one or more of brown algae such as sea brown algae including herba Zosterae Marinae, thallus laminariae, Sargassum, sea oak, cornu Cervi, Ascophyllum nodosum, thallus laminariae, Cladosiphon okamuranus, Sargassum thunbergii, Sargassum integrifolium, Cyrtymenia Sparsa, Sargassum salmias, semen Panici Miliacei, and semen Artemisiae Annuae.
The intestinal flora regulator can improve the structure of intestinal flora, improve the relative abundance of intestinal probiotics, maintain the thickness of intestinal mucosa and increase secondary metabolites of the intestinal flora.
The further intestinal flora regulator can improve the structure of intestinal flora, specifically increase the proportion of Verrucomicrobia (Verrucomicrobia) in intestinal tract and increase the ratio of Bacteroides to firmicnobia.
Further gut flora modulators are able to increase the relative abundance of gut probiotics, in particular of the probiotics Akkermansia and/or a.
Wherein, Akkermansiaceae can prevent weight gain caused by high fat diet, repair damaged intestinal barrier integrity, reduce endotoxin level in blood, and improve insulin resistance. Oral Akkermansia was shown to reverse the metabolic syndrome induced by high fat diet in mice, suggesting a potential approach to treat metabolic syndrome by manipulating Akkermansia abundance in the gut flora. Muciniphila is a widely regarded intestinal probiotic. The specific degradation of mucin makes it a key microorganism for maintaining the barrier function of intestinal mucosa. The A.muciniphila in intestinal tract can protect intestinal barrier function, reduce endotoxin level in blood plasma, and improve low-grade inflammation and metabolic disorder of organism.
The further intestinal flora regulator can increase the secondary metabolite of the intestinal flora, in particular to improve the generation of short-chain fatty acid in the intestinal tract. Short chain fatty acids are short chain organic acids containing less than 6 carbon atoms, particularly acetic, propionic, and butyric acids, produced by the digestion of dietary fiber by some intestinal bacteria. There is evidence that short chain fatty acids can reduce appetite and/or alter energy metabolism to promote health.
The intestinal flora regulator can reduce hyperglycemia, insulin resistance and hyperlipidemia symptoms of mice with metabolic disorder induced by high fat diet; protecting liver and kidney function, reducing hyperuricemia and non-alcoholic fatty liver, and reducing inflammatory reaction of organism.
The intestinal flora regulator of the invention has wide application in foods, medicines and health products for treating and preventing symptoms/diseases related to metabolic disorder.
Drawings
Figure 1. effect of fucoidan of different molecular weight on blood glucose (a) and abundance of a. muciniphila in the gut of HFD mice (b).
Fig. 2 intestinal flora modulators may improve HFD-induced intestinal microbial dysregulation. Species distribution plots, with different colors representing different phylum classification levels.
Figure 3 LEFse analyses bacterial populations with significant differences among groups.
Figure 4 level of short chain fatty acids in colon contents. Data are mean ± sd (n-8). Significance is expressed as p<0.05,**p<0.01, vs negative group;#p<0.05or##p<0.01, vs control group.
FIG. 5 intestinal flora modulators improve insulin resistance and increase insulin sensitivity in HFD mice. (a) The method comprises the following steps The intestinal flora regulator reduces the fasting blood glucose level of HFD mice; (b) the method comprises the following steps The intestinal flora regulator reduces the fasting insulin level of HFD mice; (c) the method comprises the following steps A plot of blood glucose over time; (d) the method comprises the following steps The intestinal flora regulator reduces fasting glucose tolerance of HFD mice and increases insulin 0 high fat diet; DFPS1.0-L, 100 mg/kg/d; DFPS 1.0-H200 mg/kg/d; the dose of Met to metformin is 200 mg/kg/d. Data are mean ± sd. Significance is expressed as p<0.05,**p<0.01, vs negative group;#p<0.05or##p<0.01, vs control group
FIG. 6 is a graph of the effect of gut flora modulators on the biochemical indices of the liver and kidney of HFD mice. (a) The method comprises the following steps The intestinal flora regulator reduces the liver index of the HFD mice; (b) the method comprises the following steps Effects of gut flora regulators on alanine Aminotransferase (ALT) and alanine Aminotransferase (AST); (c) the method comprises the following steps Effects of gut flora modulators on Albumin (ALB) and Globulin (GLOB). Gut flora modulators can significantly reduce urea nitrogen (urea) (d), Uric Acid (UA) (e), and Creatinine (CRE) (f) levels in HFD mice.
FIG. 7 intestinal flora modulators reduce liver cell steatosis, lipid deposition, reduce liver inflammatory response and oxidative stress. (a) Histological analysis was performed using hematoxylin and eosin staining (H & E staining). White fat vacuoles (red arrows); cell balloon-like changes (black arrows); uneven cytoplasmic staining of hepatocytes (green arrows); (b) western blot results show that the intestinal flora regulator reduces the expression of TNF alpha, IL-6 and MCP-1 and reduces the inflammatory reaction of the liver.
Detailed Description
The invention is further illustrated by the following examples, but the scope of the invention as claimed is not limited to the examples.
Example 1: regulation of blood glucose and intestinal flora in High Fat Diet (HFD) -induced mice by fucoidan of different molecular weights
Selecting male mice of C57BL/6J with kelp-derived fucoidan FL (1030Da), FM (6000Da), and FH (10kDa) of four weeks old, weighing 20 + -2 g, dividing into 4 groups of 6 mice, feeding 60% fat-powered high fat feed, and molding for 7 weeks before administration. FL (1.3Da), FM (7.2kDa), FH (10kDa) were gavaged at 200 mg/kg/day, and the blank group was gavaged with saline of the same volume. High-fat diet was continuously fed during the gavage period and blood glucose changes were monitored. After 8 weeks of dosing, mice were blood glucose measured and mouse feces were collected using sterile metabolic cages for analysis of intestinal microbes.
(1) Intestinal flora modifier for improving intestinal flora structure
The V3+ V4 region of the 16s rDNA of the enteric bacteria was amplified using primers 341F (5'-CCTAYGGGRBGCASCAG-3') and 806R (5'-GGACTACNNGGGTATCTAAT-3') (341-806). The amplification products were then pooled, purified and quantified by a fluorescence quantifier Qubit 3.0(Thermo fisher technology co., New York, USA). A new generation of sequencing was performed by Illumina Hiseq 2500PE250(Illumina, Inc, California, USA) in midio biotechnology, ltd. Bioinformatic analysis was performed using the real-time interactive data analysis online platform, Omicsmart (http:// www.omicsmart.com).
Figure 1 shows that FM and FH significantly reduced fasting blood glucose levels in HFD mice after 8 weeks of gavage, with FM showing better hypoglycemic activity than FH. Intestinal bacteria were identified and the relative abundance of probiotic a. muciniphila was found to be significantly increased in the intestines of FM group mice.
Example 2: gut flora modulator improves High Fat Diet (HFD) -induced disturbances of the gut flora in mice
The experiment was carried out using an intestinal flora regulator extracted from kelp, wherein the fucose content was 33.2%, the sulfate group content was 29.3%, and the weight average molecular weight was 7.2kDa.
Four-week-old C57BL/6J male mice were selected, and the mice were divided into 5 groups (blank group-CK group, high fat diet group-HFD group, fucoidan low dose group-DFPS-L group, fucoidan high dose group-DFPS-H group, metformin group-Met group) with a body weight of 20. + -.2 g, and 12 mice per group. The blank group was fed with 10% fat-powered normal feed. The remaining 4 groups were fed with 60% fat-energized high-fat diet, and administration was started 7 weeks after molding. The gavage normal saline is gavage 100 mg/kg/day in the high-fat diet group, the gavage 200 mg/kg/day in the high-dose group, the metformin (sigma aldrich Shanghai trade Co., Ltd.) is selected as the positive drug, and the gavage dose is 200 mg/kg/day. During the gavage period, high-fat diet was continuously fed and changes in body weight, diet, and blood sugar were monitored. After 8 weeks of dosing, mouse feces were collected in sterile metabolic cages for analysis of intestinal microbes. The mouse is dissected, and liquid nitrogen treatment is carried out on serum, liver, colon content and the like, and then the mouse is placed in a refrigerator at the temperature of-80 ℃ for standby application, and the mouse is used for biochemical determination and tissue morphology observation.
(1) Intestinal flora modifier for improving intestinal flora structure
The V3+ V4 region of the 16s rDNA of the enteric bacteria was amplified using primers 341F (5'-CCTAYGGGRBGCASCAG-3') and 806R (5'-GGACTACNNGGGTATCTAAT-3') (341-806). The amplification products were then pooled, purified and quantified by a fluorescence quantifier Qubit 3.0(Thermo fisher technology co., New York, USA). A new generation of sequencing was performed by Illumina Hiseq 2500PE250(Illumina, Inc, California, USA) in midio biotechnology, ltd. Bioinformatic analysis was performed using the real-time interactive data analysis online platform, Omicsmart (http:// www.omicsmart.com).
Fig. 1 shows that the ratio of Firmicutes to bacteroides in the HFD group is reversed (HFD: CK 0.18/2.95) and the relative abundance of Proteobacteria is significantly higher than that in the CK group (1.98%) by 13.62%. And proteobacteria are the hallmark of intestinal dysbacteriosis. DFPS significantly reduced the proportion of proteobacteria, especially in the high dose group (2.95%). The firmicutes of the DFPS group were dose-dependent decreased from 23.02% to 16.97% compared to HFD mice (61.73%). The changes described above result in a reduction in the proportion of both phyla (Firmicutes and bacterioides) in the overall gut microbiota. The relative abundance of Verrucomicrobia in HFD mice (1.92%) was significantly lower than that of CK group (15.28%). DFPS, in turn, increased the proportion of Microwartia bacteria from 40.93% to 67.65% and was dose-dependent. These changes are similar to the effect of metformin on gut microbiota.
(2) Intestinal flora regulator for increasing content of intestinal probiotics
LEFse analyzed groups of bacteria with significant differences among groups. Compared to the HFD mouse intestinal flora, the addition of DFPS and the addition of metformin showed similar differences in specific genera, with Verrucomicrobia specificity increased. The intestinal microorganism, Akkermansiaceae, which is the most representative of Verrucomicrobia, was significantly elevated in the intestine of mice supplemented with DFPS and metformin. In addition, Akkermansia _ muciniphila outbreaks also occurred in the high dose group and the metformin group. DFPS predisposes the gut flora structure to metformin on the gut level as well as on species composition. Functional prediction of picrusst 2 shows that DFPS significantly reduces the metabolic levels of three nutrients (carbohydrate, fat and protein), reducing the energy metabolism level of the body.
(3) Intestinal flora modulator for increasing production of short chain fatty acids
The colon contents (25mg) were suspended in 1.5mL of water and adjusted to pH 2-3 with 1M HCl solution. Ultrasonic extraction was carried out in an ice bath for 20 minutes, and centrifugation was carried out at 12000rpm for 15 minutes. The supernatant was added with 1ml of ethyl acetate, centrifuged at 12000rpm for 15min by vortexing, and the ethyl acetate layer was subjected to gas chromatography (Agilent 7890a, Agilent Technologies, Ca, USA). SCFAs standards were purchased from Sigma (Shanghai Sigma Aldrich training co., Shanghai, China).
Short chain fatty acids are short chain organic acids, containing less than six carbon atoms, produced by the digestion of dietary fiber by some intestinal bacteria. The effect of DFPS on intestinal flora has led us to focus on its effect on short chain fatty acids. Figure 3 shows that DFPS significantly increased intestinal short chain fatty acid levels in both low and high dose groups.
Example 3: intestinal flora modulators ameliorate High Fat Diet (HFD) -induced metabolic disorders in mice
At the end of week 8, mice were fasted for 12h and gavaged with 2g/kg of D-glucose solution. Blood glucose determination of mice blood glucose was determined 0, 30, 90 and 120min after gavage using a Roche Diagnostics GmbH (ACCU-CHEK Performa) glucometer (Roche Diagnostics GmbH, Basel, Switzerland). Glucose change curves were plotted and oral glucose tolerance (OGTT) was assessed by calculating the area under the curve. The steady state model evaluation index (HOMA-IR) was used to evaluate insulin resistance in mice according to the following formula:
HOMA-IR index ═ fasting blood glucose (mmol/L) × fasting insulin (mU/mL) ]/22.5.
At the end of 8 weeks, mouse serum was collected and biochemical indicators of blood lipid were determined using a fully automatic biochemical analyzer (laborpt 008AS, hitachi high-tech limited, tokyo, japan) of the china Qingdao gold domain detection center.
High Fat Diet (HFD) significantly increased fasting blood glucose and fasting insulin levels (fig. 4a, b), increased glucose tolerance, and decreased insulin sensitivity. The hypoglycemic effect of DFPS is dose-dependent. Low doses of DFPS significantly reduced fasting glucose and glucose tolerance, restored insulin sensitivity, and improved insulin resistance (fig. 4 e). The HOMA-IR model also showed that DFPS was effective in improving insulin resistance in HFD mice (FIG. 4 f). In particular, large doses of DFPS have the same hypoglycemic effect as metformin.
Dyslipidemia is an important feature of metabolic syndrome, including elevation of TC, CHOL, HDL-C and lowering of HDL-C. Table 1 shows that HFD causes significant increases in TG, CHOL and LDL-C and abnormal increases in HDL-C. The low dose of DFPS significantly reduced CHOL, LDL-C and HDL-C levels, and the high dose significantly reduced TG levels. The reduction of Free Fatty Acids (FFA) by DFPS was not significant. Among them, DFPS is superior to metformin in lowering CHOL, LDL-C and HDL-C, suggesting that DFPS contributes to restoration of lipid homeostasis.
Table 1. biochemical index of blood fat
And Note is biochemical index of blood fat. TG, CHOL, LDL-C, HDL-C, TG, CHOL, LDL-C, HDL-C, Free Fatty Acids (FFA) and Visceral Fat Index (VFI). Data are presented as mean ± SD (n ═ 8).abcGroups that are not identical letters in a row are indicated as significantly different groups, at a level of p < 0.05 by one-way ANOVA test.
Example 4: intestinal flora regulator for improving High Fat Diet (HFD) -induced liver and kidney function injury of mice
A full-automatic biochemical analyzer (LABOSPECT 008AS, Hitachi high-tech limited company, Tokyo, Japan) of the detection center of Qingdao gold field in China is adopted to measure biochemical indexes of liver function and kidney function.
The liver is the largest organ of the body and is the central organ that regulates lipid metabolism. Dyslipidemia and accumulation of hepatic triglycerides cause liver disease, thereby affecting the normal function of the liver. The biochemical index of the serum liver can reflect the pathological changes of the liver. DFPS reduced liver lipid accumulation, manifested as a decrease in liver index (fig. 5 a). ALT and AST are sensitive indicators of liver cell damage. When cells are damaged and cell membrane permeability increases, the levels of both enzymes in serum rise. As shown in fig. 5b, HFD doubled ALT and AST levels and significant damage to hepatocytes. DFPS significantly reduced AST levels, but low doses had no significant effect on ALT levels. At high doses, levels of alanine Aminotransferase (ALT) and aspartate Aminotransferase (AST) were both significantly reduced, suggesting that DFPS may reduce hepatocellular injury. In addition, DFPS also significantly reduced GLOB, suggesting that DFPS may reduce liver inflammation and infection (fig. 5 c). In conclusion, supplementation with DFPS can ameliorate the metabolic dysfunction caused by HFD. The protective effect of DFPS on the kidney of HFD mice was further observed. DFPS significantly reduced UA, CRE and urea even to normal levels (fig. 5d, e, f), indicating that DFPS reduced the incidence of renal insufficiency and hyperuricemia in HFD mice.
Fresh liver tissue was taken for histological analysis. The tissue was soaked in 4% paraformaldehyde for 24h, dehydrated and embedded in paraffin. The fixed tissues were cut into 5 μm thick sections and stained with hematoxylin and eosin (H & E). The slices were scanned by a digital pathology scanner (Leica Aperio CS2, Leica biological Systems inc., Nussloch, Germany) to generate representative images.
The effect of DFPS on liver was further analyzed by histological analysis. As can be seen in fig. 6a, HFD increases liver fat deposition, altering liver morphology. The hepatic lobular margin of HFD mice was unclear. Intrahepatic fat deposits form white fat vacuoles (red arrows) and intrahepatic fat pushes the nucleus to the edge, making the hepatocytes balloon-like (black arrows). Meanwhile, uneven cytoplasmic staining of hepatocytes (green arrows) may indicate hepatocyte deformation or necrosis. DFPS reduced the number of fat vacuoles and no significant cell deformation or necrosis was seen. In particular, high doses of DFPS can even restore liver morphology to normal levels. Histological observation shows that DFPS can effectively reduce liver fat deposition and reduce liver cell pathological changes caused by HFD.
Extracting the hepatic protein to carry out western blot. Liver tissue (20mg) was washed twice with cold PBS and lysis buffer (containing 0.1mM PMSF and protease inhibitor) was dissolved. Protein concentration was determined using BCA kit (Solaibao Technology co., Beijing, China). Proteins were separated by polyacrylamide gel electrophoresis (SDS-PAGE), transferred to PVDF membrane, blotted with each antibody, and detected with ECL reagent. Tumor necrosis factor-alpha (TNF-alpha), monocyte chemoattractant protein 1(MCP-1), and interleukin-6 (IL-6) (Santa Cruz Biotechnology, Santa Cruz, Calif., USA) were determined.
It is well known that chronic inflammation plays an important role in the development of T2DM and MetS 21. Accumulation of liver fat leads to an increase in inflammatory cytokines and oxidative stress. Western blot was used to detect the expression of inflammatory cytokines (TNF. alpha., IL-6, MCP-1) in liver tissues. As shown in fig. 6b, DFPS effectively reduced inflammatory factor levels, attenuating HFD-induced inflammatory responses. The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof; such modifications and substitutions do not depart from the spirit and scope of the corresponding technical solutions.