Use of fermented cereals in the manufacture of products for alleviating the damage caused by smoking

Abstract

The application discloses application of fermented grains in preparation of products for reducing body damage caused by smoking (including active and passive smoking); and the fermentation bacteria of the fermented grains are mainly lactobacillus plantarum. The application is used for reducing reproductive organ and lung damage caused by smoking, and has the technical effect of significantly improving metabolism and intestinal flora disorder.

Description

TECHNICAL FIELD

[0001] The present application relates to the application of fermented cereals in the preparation of products for reducing the damage to the body caused by smoking, and in particular to the application of Lactobacillus plantarum fermented whole cereals in the preparation of products for reducing the damage to the body caused by active and passive smoking, metabolic abnormalities and intestinal flora disorders. BACKGROUND

[0002] According to statistics, 22% of the global population over the age of 15 smoke, and about 6 million people die each year due to smoking-related diseases, and tobacco-related deaths have become the first cause of death in the world [WHO. World health statistics 2012[R]. Geneva: Switzerland, 2012]. China is a recognized large producer and consumer of tobacco, and tobacco production accounts for more than 40% of global tobacco production [Eriksen M, Mackay J, Ross H. American cancer society and world lung foundation [EB / OL]. http: / / www.Tobaccoatlas.org / .[2014-10-07]], and the number of smokers in China currently reaches 350 million, of which 52.9% are male, and in addition, smoking also causes about 740 million non-smokers to be exposed to second-hand smoke in public places, and it is estimated that the number of annual deaths due to tobacco use in China will exceed 3 million by 2050 [Yang G, Wang Y, Zeng Y, et al. Rapid health transition in China, 1990-2010: findings from the Global Burden of Disease Study 2010 [J]. Lancet, 2013, 381: 1987-2015.].

[0003] Smoking is considered to be closely related to the occurrence and development of various diseases such as cancer, cardiovascular lesions and respiratory damage.

[0004] Firstly, for the reproductive system, studies have shown that smoking affects the quality of male semen [Toshima H, Suzuki Y, Imai K, et al. Endocrine disrupting chemicals in urine of Japanese male partners of subfertile couples: a pilot study on exposure and semen quality [J]. Int J Hyg Environ Health, 2011, 215: 502-506.]. The testis is one of the target organs of the toxic effects of cigarette smoke, and cigarette smoke has toxic effects on the testis of rats and affects its growth and development and normal reproductive function. Belcheva et al. found that new chemical substances produced by cigarette smoke can affect the growth and development of rapidly dividing sperm cells, and DNA damage in sperm is exacerbated after exposure to tobacco smoke [Belcheva A, Ivanova kicheva M, Tzvetkova P, et al. Effects of cigarette smoking on sperm plasma membrane integrity and DNA fragmentation [J]. International Journal of Andrology, 2010, 27(5): 296-300.]; Zhang Jing et al. showed that passive smoking of cigarette smoke can cause changes in the morphology of mouse sperm [Zhang Jing, Deng Sheng, Huang Yunfei, et al. Effects of tobacco smoke on the morphology of mouse sperm and some biochemical indicators of the testis [J]. Journal of Toxicology, 2014, 28(2): 117-119.]. Studies have shown that smoking can cause damage to the reproductive system and affect reproductive capacity.

[0005] Secondly, in terms of body metabolism, smoking and physical inactivity are risk factors for hypertension, diabetes, and dyslipidemia in adult men. Now, the risk of hypertension, diabetes, and dyslipidemia is higher in people who smoke and are physically inactive [Wu J, Yang HF, Qi SX, Wang CC, Zhou H, Qin ZZ, Li C, Ye Q, Wang WW, Hong X. Association between smoking and physical inactivity and hypertension, diabetes, and dyslipidemia in adult men. Chinese Journal of Health Management. 2021, 15(02): 138-143]. Many studies have shown that smoking increases the risk of metabolic syndrome [Geslain-Biquez C, Vol S, Tichet J, et al. The metabolic syndrome in smokers. The DESIR study [J]. Diabetes & Metabolism, 2003, 29(3): 226-234], Nakanisi et al. Prospective study of smoking population found that the risk of developing metabolic syndrome in smokers was 1.07-1.66 times that of non-smokers [Nakanishi N, Takatorige T, Suzuki K. Cigarette smoking and the risk of the metabolic syndrome in middle-aged Japanese male office workers [J]. Industrial Health, 2005, 43(2): 295-301], one of the manifestations of metabolic syndrome is glucose and lipid metabolism disorder, this pathological state also includes insulin resistance, hypertension, coagulation system abnormalities and a series of metabolic disorders signs, it can be seen that smoking can cause abnormal metabolism of the body.

[0006] Finally, in terms of intestinal microecology, some studies have shown that smoking can change the composition of intestinal microorganisms, including the increase of Proteobacteria and Bacteroidetes phylum, Clostridium, Bacteroides and Prevotella genus, and the decrease of Actinobacteria, Firmicutes, and Bifidobacterium and Lactococcus genus; Smoking also causes a decrease in intestinal flora diversity [Savin Z, Kivity S, Yonath H, Yehuda S. Smoking and the intestinal microbiome. Arch Microbiol. 2018 Jul; 200(5): 677-684.], changes in intestinal flora can cause changes in intestinal metabolic products, further affecting the metabolism of the body.

[0007] It is known that smoking can cause harm to the body in many aspects, such as reproduction, metabolism and intestinal microenvironment. In addition to smoking cessation, how to reduce the damage of smoking to the body through dietary intervention is worth paying attention to, but there are few relevant systematic studies.

[0008] The prior art CN 103813798 A obtains a polysaccharide polymer from mangrove seeds, which is used to prevent the damage of tobacco to respiratory epithelial cells, and the main administration mode is an aerosol delivery system. The disadvantages of this technology are that the protection agent administration process needs to use a commercially available sprayer, which is inconvenient to implement; and the functional detection method used is an in vitro cell experiment, the application effect in vivo is not determined; more importantly, the protection effect of the polysaccharide polymer on tobacco is only the respiratory system epithelial cells, and the intervention effect on multiple target organs and the whole body of the body lacks effective data support. SUMMARY

[0009] In view of the above limitations of the prior art, the technical problem to be solved by the present application is how to utilize fermented black barley to provide a new use: application of a fermented cereal in preparation of a product for reducing damage to the body (reproductive system, metabolism and intestinal microenvironment) caused by smoking.

[0010] To achieve the above-mentioned purpose, the present application provides a fermented cereal, the raw material of which is single or combined cereal of black barley, brown rice and quinoa; the fermentation strain used is Lactobacillus kisonensis, or mixed fermentation with other lactic acid bacteria.

[0011] The present application also provides application of a fermented cereal in preparation of a product for reducing damage to the body and metabolism caused by smoking.

[0012] The present application also provides application of a fermented cereal in preparation of a product for reducing damage to tissues and organs caused by smoking, which includes heart, kidney, liver and lung.

[0013] The present application also provides application of a fermented cereal in preparation of a product for reducing damage to the reproductive system caused by smoking.

[0014] The present application also provides application of a fermented cereal in preparation of a product for reducing intestinal flora disorder caused by smoking.

[0015] The smoking includes active smoking and / or passive smoking.

[0016] As an embodiment of the present application, the product is a fermented cereal drink, a solid powdered fermented cereal, a fermented cereal capsule, a fermented cereal tablet or a fermented cereal pill. The solid powdered fermented cereal is processed by freeze-drying or spray-drying technology.

[0017] As an embodiment of the present application, the product is a functional food, and the method of using the product is oral administration.

[0018] In some embodiments, the product is a functional food prepared by using the fermented cereal as the main active ingredient, and fruit and vegetable powder and / or probiotics.

[0019] In some embodiments, the functional food includes a drink or a solid beverage to be dissolved, and the dosage form of the functional food is a capsule, a tablet, a pill, a granule or a suspension.

[0020] The method of using the product includes directly orally taking a fermented cereal drink, a fermented cereal powder to be dissolved, or a functional food prepared by using the fermented cereal as the main ingredient, and fruit and vegetable powder and / or probiotics.

[0021] As an embodiment of the present application, the fermented cereal is prepared by a method comprising the following steps:

[0022] S1, crushing the single or combined cereal and passing through a 60-80 mesh sieve, adding pure water at a mass ratio of 1:5-1:7, and stirring and heating to 63-67°C;

[0023] S2, adding α-high temperature amylase at an addition amount of 9.5-10.5 U / g, and enzymolysis for 35-45 minutes, to obtain a whole cereal culture medium after the enzymolysis is completed;

[0024] S3, sterilizing the cereal culture medium, adding activated fermentation strain bacteria liquid at an inoculation amount of 2-5% after cooling, and fermenting at 25-35°C for 24-40 hours at a shaking speed of 90-110 rpm.

[0025] As an embodiment of the present application, in step S3, the activated fermentation strain bacteria liquid contains 10 9 CFU / ml of viable bacteria, and the viable bacteria number is 10 8 CFU / ml at the end of fermentation.

[0026] Compared with the prior art, the present application has the following beneficial effects:

[0027] 1) The lactobacillus fermented black barley provided by the present application can significantly reduce the damage to the reproductive organs and lungs caused by active or passive smoking, and can improve the metabolic abnormalities and intestinal flora disorders caused by smoking.

[0028] 2) The lactobacillus fermented black barley provided by the present application has no adverse effects on animal growth and other aspects.

[0029] 3) The fermented black malt provided by the present application can be used as a functional food for preventing and reducing the damage to the body caused by smoking, or as a main component of a composition combined with related components, and has a good application prospect in combination with the current epidemiological investigation of the smoking population;

[0030] 4) The fermented black malt can reduce the damage of smoking to the tissues and organs of mice, including that the fermented black malt intervention can effectively improve the morphological changes of the heart caused by smoking, the liver / body ratio of the mice in the fermented black malt intervention group is lower than that of the mice in the smoking group alone, and the kidney index of the mice after the fermented black malt intervention is lower than that of the mice in the smoking group alone;

[0031] 5) Effects of the fermented black malt on the reproductive system of the smoking mice: the fermented black malt intervention can reduce the changes in the size and shape of the testis caused by smoking; the total sperm motility of the mice in the fermented black malt intervention group is increased by 31.99% (p<0.05) compared with that of the mice in the smoking group alone, and has no significant difference (p>0.05) with that of the normal control group; after the fermented black malt intervention, the testis of the mice is significantly increased (p<0.05) compared with that of the mice in the smoking group alone, and has no significant difference (p>0.05) with that of the normal control group; the number of germ cells in the testis of the mice in the fermented black malt intervention group is increased, sperm formation can be observed in the lumen, the number of seminiferous tubules is more, and the connective tissue between the seminiferous tubules is normal;

[0032] 6) Effects of the fermented black malt on the antioxidant status of the lung tissue of the smoking mice: the four indexes of CAT, GSH-Px, GSH and SOD of the lung tissue of the mice in the fermented black malt intervention group are increased by 10.97%, 29.63%, 84.35% and 10.10% (p<0.05) compared with those of the mice in the smoking group alone, and the GSH has no significant difference with that of the normal control group; the MDA level of the lung tissue of the mice in the fermented black malt intervention group is decreased by 16.33% (p<0.001) compared with that of the mice in the smoking group alone; it is shown that the fermented black malt intervention can enhance the antioxidant capacity of the lung tissue of the smoking mice and reduce the oxidative stress damage;

[0033] 7) Effects of the fermented black malt on the pathological morphology of the lung tissue of the smoking mice: the bronchus, alveolar duct, alveolar capsule and alveolus structures of the lung tissue of the mice in the fermented black malt intervention group can be identified, the bronchus wall is slightly damaged, the alveolus can be observed, the alveolar septum structure is relatively complete, and the inflammatory cell infiltration phenomenon is slightly relieved compared with that of the mice in the smoking group alone. The results show that the fermented black malt intervention has a good protective effect on the integrity of the bronchus, alveolus and other structures of the lung tissue;

[0034] 8) The effect of fermented black malt on the intestinal flora of smoking mice: The Shannon index and Simpson index of the fecal flora of mice in the fermented black malt intervention group were not significantly different from those of the normal control group (p>0.05), and were higher than those of the smoking group alone; fermented black malt intervention can significantly reduce the relative abundance of Actinobacteria, and has no significant difference with the normal control group; in addition, fermented black malt intervention can significantly increase the relative abundance of three bacterial phyla, Tenericutes (p<0.01), TM7 (p<0.05) and Deferribacteres (p<0.05) compared with the smoking group; at the genus level, the flora distribution of the fermented black malt intervention group is closer to that of the normal control group;

[0035] 9) The effect of fermented black malt on the liver and fecal metabolites of smoking mice: In positive and negative ion modes, the fermented black malt intervention group and the smoking mouse group are completely distinguished, and both are close to the normal control group, so fermented black malt intervention can effectively improve the abnormal liver metabolism caused by smoking; In particular, among the liver differential metabolites, smoking can significantly reduce 13 substances such as p-Cymene, Estradiol, Nandrolone, Neriifolin, etc., while fermented black malt intervention can significantly increase the above substances. Among them, Nandrolone and Estradiol are steroid substances that play a role as sex hormones in the body, which has certain relevance to the protection of the reproductive system of smoking mice by fermented black malt; In addition, fermented black malt intervention can also alleviate the impact of smoking on fecal metabolites in mice to a certain extent, and play a protective role in the intestinal microenvironment. BRIEF DESCRIPTION OF DRAWINGS

[0036] Other features, objects, and advantages of the application will become more apparent from the following detailed description of non-limiting embodiments, made with reference to the following drawings:

[0037] Figure 1 The effect of fermented black malt on the indices of some organs (heart, liver, kidney) of smoking mice;

[0038] Figure 2 The effect of fermented black malt on the reproductive system of smoking mice; Wherein, A is the comparison of the testis weight of mice in each group; B is the comparison and analysis of the total sperm motility of mice in each group; C is the comparison of the testis appearance of the three groups of mice; D is the HE staining result of the testis tissue section of each group of mice;

[0039] Figure 3 The effect of fermented black malt on the antioxidant index of lung tissue of smoking mice;

[0040] Figure 4 The effect of fermented black malt on the pathological morphology of lung tissue of smoking mice;

[0041] Figure 5 Effects of fermented black malt on intestinal flora diversity of smoking mice; wherein A is a preliminary evaluation using Chao1 index; B is a preliminary evaluation using Shannon index; C is a preliminary evaluation using Simpson index;

[0042] Figure 6 Effects of fermented black malt on intestinal flora distribution of smoking mice; wherein A is a comparison of the relative abundance of each group of mice fecal flora at the door level; B is a cluster analysis using a heat map of the top 20 relative abundance of each group of mice at the genus level;

[0043] Figure 7 PLS-DA cluster analysis chart of mouse liver metabolites under negative ion mode (A) and positive ion mode (B);

[0044] Figure 8 Effects of fermented black malt on fecal metabolites of smoking mice;

[0045] Figure 9 PLS-DA cluster analysis chart of mouse fecal differential metabolites under negative ion mode (A) and positive ion mode (B). DETAILED DESCRIPTION

[0046] The present application will be described in detail below with reference to the examples. The following examples will help those skilled in the art to further understand the present application, but do not limit the present application in any form. It should be noted that for those skilled in the art, without departing from the concept of the present application, a number of adjustments and improvements can be made. These are within the scope of the present application.

[0047] Example 1

[0048] Preparation of lactobacillus fermented black malt:

[0049] The black malt was crushed and passed through an 80 mesh sieve, and then water was added at a solid-liquid ratio of 1:5 (m / m) and stirred and heated to 65°C. α-high temperature amylase (10 U / g) was added and enzyme hydrolysis was carried out for 40 min. The black malt medium obtained after enzyme hydrolysis was sterilized in a high-pressure sterilization pot, and after cooling, activated lactobacillus plantarum liquid (containing 109CFU / ml of viable bacteria) was added to the cooled black malt medium at an inoculation amount of 3%, and fermentation was carried out at 30°C for 24 h with a shaking bed (100 rpm).

[0050] Lactobacillus fermented black malt and its fermentation strain as a health product (functional food) for reducing smoking-induced lung damage and intestinal flora disorder. The following are the main food functional tests:

[0051] 1. Animal experiment intervention method and index analysis method

[0052] In order to observe the effect of lactobacillus fermented black malt intervention on the body caused by smoking, 6-week-old male clean ICR mice (30±2.0g) were purchased from Shanghai Slek Experimental Animal Co., Ltd. After one week of adaptive culture, the mice were randomly divided into three groups according to body weight: normal control group (NC, n=7), smoking group (S, n=7), and fermented black malt intervention group (SB, n=7). The mice in the smoking group and the fermented black malt group were exposed to tobacco smoke daily at a fixed time (self-made cigarette exposure box), with each exposure lasting 30 minutes and the smoke dose being 15 cigarettes. The fermented black malt intervention group was given gavage treatment at a fixed time every day, with the gavage dose being 100 μL / 10g·body weight (BW) (equivalent to 1 mL / kg·BW in humans). The intervention lasted for 12 weeks, during which the daily food intake of the mice and the body weight changes of the mice every half month were recorded. At the end of the experiment, the mice were sacrificed by cervical dislocation under anesthesia, and the lung and other tissues were taken for analysis and detection as follows.

[0053] (1) Organ index: heart index, liver index, kidney and testis index, the body weight of the mice before sacrifice was measured. The testis, heart, liver and both kidneys of the mice were carefully removed at sacrifice, washed with normal saline, and the excess water was absorbed with clean filter paper. The mass of each tissue organ was accurately measured. Heart index (%) = heart mass / mouse mass*100; liver index (%) = liver mass / mouse mass*100; kidney index (%) = both kidney mass / mouse mass*100.

[0054] (2) Preparation of testis and lung tissue pathological sections: fresh testis and lung tissue was fixed, paraffin-embedded, and pathological sections were prepared by HE staining method, and observed under light microscope.

[0055] (3) Total sperm motility determination: the intact testis tissue was weighed, and one side of the testis was cut into several pieces with ophthalmic scissors in culture medium. The testis tissue was squeezed to make sperm enter the sperm capacitation liquid, and the tissue pieces were discarded. The sperm culture medium was incubated in an incubator to fully disperse the sperm. The mixed and uniform culture medium was dropped onto the detection plate, and the SSA-II sperm automatic detection analyzer was used to determine the total sperm motility of the mice.

[0056] (4) Lung tissue antioxidant capacity determination: 0.02 g of lung tissue was accurately weighed, and physiological saline was added at a ratio of 1:10 (w / v). The tissue homogenate was prepared using a mechanical homogenizer under ice water bath conditions. Centrifugation was performed at 2500 rpm / min for 10 minutes, and the supernatant was taken. The CAT, GSH-PX, MDA, and SOD four antioxidant indexes were determined and calculated according to the kit instructions. 0.01 g of lung tissue was accurately weighed, and GSH reagent one was added at a ratio of 1:10 (w / v). The tissue homogenate was prepared using a mechanical homogenizer under ice water bath conditions. Centrifugation was performed at 2500 rpm / min for 10 minutes, and the supernatant was taken. The GSH index was determined and calculated according to the kit instructions.

[0057] (5) Intestinal flora analysis: 1-2 weeks before the end of the experiment, the mouse feces were collected aseptically, and the diversity of mouse intestinal flora and the relative abundance distribution characteristics of intestinal flora in each group of mice at the door, class, order, family, genus, and species were analyzed by 16S rDNA sequencing.

[0058] (6) Differential metabolite analysis: non-target UPLC-Q-TOF-MS E was used to analyze the abundance of small molecule differential metabolites in the liver and feces of each group of mice.

[0059] 2. Data processing

[0060] SPSS 24 (IBM, USA) software was used, and the single factor analysis of variance (One-way ANOVA) method was used for significant analysis of the differences between groups, and the LSD method was used to compare the differences between the model group and the other two groups. GraphPad Prism 8.0 (Graphpad Software, USA) software was used to draw images.

[0061] 3. Functional experiment results

[0062] The experimental results show that lactobacillus fermented black barley can significantly reduce the enlargement of some organs caused by smoking, improve the decline of reproductive system function and intestinal flora disorder caused by smoking, and correct the abnormal metabolism of the body. The main functional experiment results are described as follows:

[0063] (1) Fermented black barley can reduce the damage of smoking to mouse tissues and organs

[0064] As Figure 1 shown, lactobacillus fermented black barley has an effect on the index of some organs of smoking mice. From Figure 1It can be seen that compared with the NC group, the heart index of the smoking group mice was increased, and the average heart index of the smoking group mice (S group) was 0.629, which was increased by 25.55% (p<0.05) compared with the NC group. The heart index of the fermented black barley intervention group mice was significantly lower than that of the S group (p<0.05), and had no significant difference with the NC group (p>0.05), indicating that fermented black barley intervention can effectively improve the morphological changes of the heart caused by cigarette pollution. There was no significant difference in liver index among the three groups (p>0.05), but overall, the liver / body ratio of the cigarette pollution group was higher than that of the NC group and the SB group. In terms of kidney index, the kidney index of the smoking group mice was significantly increased compared with the NC group (p<0.01), and the kidney index of the fermented black barley intervention group mice was lower than that of the S group mice.

[0065] (2) Effect of fermented black barley on the reproductive system of smoking mice

[0066] Figure 2 A is the appearance of the testis of the three groups of mice. It can be seen that the testis of the NC group mice is uniform in shape, and is spherical; the testis of the smoking group mice is irregular in shape, and is mostly ellipsoidal, and is slightly smaller in appearance than the NC group, indicating that smoking affects the development of the testis of mice; the testis of the fermented black barley intervention group mice is uniform in shape and is spherical, and is similar in size to the NC group, indicating that fermented black barley intervention can reduce the change in size and shape of the testis caused by smoking. Figure 2 B shows the comparison and analysis of the total sperm motility of the mice in each group. Compared with the normal control group, the total sperm motility of the smoking group mice was significantly decreased by 48.08% (p<0.01); the total sperm motility of the fermented black barley intervention group mice was significantly increased by 31.99% (p<0.05) compared with the smoking group, and had no significant difference with the normal control group (p>0.05). In addition, from Figure 2 C can be seen that the testis weight of the smoking group mice was significantly decreased compared with the normal group mice (p<0.05), and after fermented black barley intervention, the testis of the mice was significantly increased compared with the smoking group (p<0.05), and had no significant difference with the normal control group (p>0.05).

[0067] Figure 2D shows the HE staining results of testicular tissue sections of mice in each group. As can be seen from the figure, the testis of the NC group is normal in shape, the cells are relatively close, the spermatogenic cells of all levels are complete and arranged in order, the sperm morphology is normal, the seminiferous tubules are complete, and the connective tissue between the seminiferous tubules is normal in shape; the testicular cell structure of the smoking group mice is relatively loose, the number of spermatogenic cells is small and the number of layers is reduced, and only spermatogonia can be observed, no sperm can be observed in the lumen, the number of seminiferous tubules is small and in a reticular shape, and calcification phenomenon occurs; the number of layers of spermatogenic cells of the fermented black barley intervention group mice increases, sperm formation can be observed in the lumen, the number of seminiferous tubules is relatively large, and the connective tissue between the seminiferous tubules is relatively normal in shape. The above results show that cigarette smoke has a great impact on the testis of mice, causing the testis to become smaller and the shape to change, the number of layers of spermatogenic cells to decrease, the production of sperm to be greatly affected, the number of seminiferous tubules to decrease and the morphological structure to change, and calcification phenomenon to occur; while fermented black barley intervention can reduce the change of the size and shape of the testis of mice caused by smoking, increase the number of layers of spermatogenic cells, produce sperm, and increase the number of seminiferous tubules and reduce morphological changes. The above results show that fermented black barley intervention has a certain protective effect on the reproductive system of smoking mice.

[0068] (3) Effect of fermented black barley on the antioxidant status of the lung tissue of smoking mice

[0069] The antioxidant indexes of the lung tissue of mice in each group, such as catalase (CAT), glutathione peroxidase (GSH-Px), malondialdehyde (MDA), glutathione (GSH) and superoxide dismutase (SOD), were also detected. The results are shown in Table 2. Figure 3 Compared with the NC group, the four indexes of CAT, GSH-Px, GSH and SOD of the lung tissue of the smoking group mice decreased by 22.88%, 40.11%, 41.62% and 23.71% respectively, all with significant differences (p<0.01), indicating that smoking can weaken the ability of mice to remove oxygen free radicals in the lung tissue, causing oxidative stress damage; the four indexes of CAT, GSH-Px, GSH and SOD of the lung tissue of the fermented black barley intervention group mice were significantly higher than those of the smoking group mice by 10.97%, 29.63%, 84.35% and 10.10% respectively (p<0.05), among which GSH had no significant difference with the normal control group. In addition, the MDA level of the lung tissue of the fermented black barley intervention group mice was significantly lower than that of the smoking group by 16.33% (p<0.001). This shows that fermented black barley intervention can enhance the antioxidant capacity of the lung tissue of smoking mice and reduce oxidative stress damage.

[0070] (4) Effect of fermented black barley on the pathological morphology of the lung tissue of smoking mice

[0071] The HE staining results of the lung tissue sections of mice in each group are shown in Table 3. Figure 4The structures of bronchus, alveolar duct, alveolar sac, alveolus and so on in the lung tissue of the mice in the NC group can be clearly distinguished, the bronchial wall is complete, there is no inflammatory cell infiltration, the alveolus is complete and easy to observe, and the alveolar septum is clearly visible. The lung abscess phenomenon in the lung tissue of the mice in the simple smoking group is serious, the bronchus, alveolar duct and alveolar sac are not clear, the alveolar structure is deformed, the bronchial wall is completely destroyed, a large number of inflammatory cells such as lymphocytes, macrophages and neutrophils are gathered, and there is a bleeding phenomenon. The structures of bronchus, alveolar duct, alveolar sac and alveolus in the lung tissue of the mice in the fermented black barley intervention group can be distinguished, the bronchial wall is slightly damaged, the alveolus can be observed, the alveolar septum structure is relatively complete, and the inflammatory cell infiltration phenomenon is slightly relieved compared with the simple smoking group. The results show that the fermented black barley intervention has a good protective effect on the integrity of the bronchus, alveolus and other structures of the lung tissue.

[0072] (5) Effect of fermented black barley on intestinal flora diversity of smoking mice

[0073] In the alpha diversity analysis, Chao1 index, Shannon index and Simpson index were used to preliminarily evaluate the species diversity of intestinal flora. Chao1 index can evaluate the species richness, representing the total number of species; Shannon index and Simpson index can evaluate the diversity and uniformity of intestinal flora. Figure 5 The results of alpha diversity analysis of intestinal flora of the three groups of mice are shown in Figure 2. Compared with the NC group, the Shannon index (B) and Simpson index (C) of the intestinal flora of the simple smoking group of mice were significantly reduced (p<0.05), while the Chao1 index (A) had no significant effect (p<0.05). The Shannon index and Simpson index of the fecal flora of the fermented black barley intervention group of mice had no significant difference compared with the NC group (p>0.05), and increased compared with the S group.

[0074] (6) Effect of fermented black barley on the distribution of intestinal flora of smoking mice

[0075] Figure 6 A is the comparison of the relative abundance of the fecal flora of the mice in each group at the phylum level. It can be seen that the relative abundance of the two bacterial phyla Firmicutes and Bacteroidetes has absolute advantage, but there is no significant difference among the three groups. The phylum Actinobacteria has significant difference between the simple smoking group and the normal control group (p<0.05). After fermented black barley intervention, the relative abundance of Actinobacteria decreased significantly, and had no significant difference with the NC group. In addition, fermented black barley intervention can significantly increase the relative abundance of Tenericutes (p<0.01), TM7 (p<0.05) and Deferribacteres (p<0.05) compared with the smoking group.

[0076] The top 20 bacteria at the genus level were clustered using a heat map, and the results are shown in FIG. 6A and FIG. 6B. Figure 6 The clustering results showed that the samples were divided into two groups. The first group contained all the samples of the smoking group and two samples of the normal control group (NC1 and NC6), and the second group contained the remaining five samples of the normal control group and all the samples of the fermented black malt intervention group. This also indicated that the distribution of the bacterial flora of the SB group was closer to that of the NC group at the genus level.

[0077] (7) Effect of fermented black malt on liver and fecal metabolites of smoking mice

[0078] The small molecule metabolites in the liver of the mice were analyzed using non-targeted UPLC-Q-TOF-MSE technology. Peak alignment, normalization, and database searching were performed on the data, and a total of 1093 substances were identified, including 288 in negative ion mode and 805 in positive ion mode. A total of 297 differential metabolites were screened out by p<0.05 and VIP>1, including 77 in negative ion mode and 220 in positive ion mode.

[0079] Figure 7 The PLS-DA clustering analysis of the liver metabolites of the mice in negative ion mode (A) and positive ion mode (B) is shown in FIG. 8. The results showed that the samples in each group were well clustered with small differences. The NC group and the S group were clearly distinguished, indicating that smoking had a certain effect on the small molecule metabolites in the liver. In both positive and negative ion modes, the fermented black malt intervention group and the S group were completely distinguished, and both were close to the NC group. Therefore, fermented black malt intervention can effectively improve the abnormal liver metabolism caused by smoking.

[0080] Further analysis of the liver metabolites is shown in FIG. 9. Figure 8Among the liver differential metabolites, 13 substances such as p-Cymene, Estradiol, Nandrolone, Neriifolin, etc. were significantly reduced by smoking, while the above substances were significantly increased after the intervention of fermented rye. Among them, Nandrolone and Estradiol are steroid substances, which play a role of sex hormone in vivo, and the change of sex hormone level can affect the development of reproductive system and sperm formation. Nandrolone is a testosterone derivative, and testosterone, as a representative androgen, is synthesized and secreted by testis and closely related to spermatogenesis. Studies have shown that there are beta estrogen receptors and G protein estrogen receptors in the development process of Sertoli cells in male rats, which can be regulated by Estradiol, so the dynamic process of spermatogenesis, including the separation and recombination of tight junctions by N-cadherin in Sertoli cells, is partly regulated by Estradiol. In this study, the abnormal testicular development and reduced sperm motility in smoking mice may be related to the decrease of sex hormone level.

[0081] Non-targeted UPLC-Q-TOF-MSE technology was used to analyze the small molecule metabolites in mouse feces. Peak alignment, normalization and database search were performed, and a total of 2625 substances were identified, including 607 in negative ion mode and 2018 in positive ion mode. p<0.05, VIP>1 as the standard, a total of 605 differential metabolites were screened, including 151 in negative ion mode and 454 in positive ion mode.

[0082] Further PLS-DA analysis was used for multi-dimensional statistical analysis of the differential metabolites in mouse feces. Figure 9 The PLS-DA clustering analysis chart of the differential metabolites in mouse feces in negative ion mode (A) and positive ion mode (B) is shown. The results show that the differential metabolites in the feces of each group of mice have small differences within the group and good clustering. The distance coefficients of the normal control group and the simple smoking group in positive and negative ion modes are larger, and they can be distinguished significantly, indicating that smoking has a certain effect on the fecal metabolites of mice. The samples of the fermented rye intervention group are separated from the smoking group, and tend to move closer to the normal control group. In positive ion mode, the confidence ellipse of the fermented rye group and the normal control group appears partially overlapped, indicating that the intervention of fermented rye can alleviate the effect of smoking on the fecal metabolites of mice to a certain extent, and plays a protective role in the intestinal microenvironment.

[0083] In addition, it needs to be explained that: the subject group of the present application has previously clarified the function of fermented black barley on the body damage caused by oil fume from two aspects of intestinal flora and antioxidant, which is different from the present application in the following aspects: ① the reasons for causing body damage are different, the present application focuses on the damage caused by smoking to the body; the gas composition of oil fume and tobacco smoke is completely different, and the action pathway and target molecule in the body are also different; ② the protection effect on the whole body is different, the present application develops the functional characteristics of fermented cereal food from multiple levels and angles such as reproductive system, body metabolism, intestinal flora and intestinal flora metabolism spectrum; ③ the present application finds that 13 substances such as p-Cymene, Estradiol, Nandrolone and Neriifolin are significantly increased in the mice after intervention of fermented black barley compared with the simple smoking group, which is similar to the normal control group. Among them, Nandrolone and Estradiol are steroid substances, which play the role of sex hormones in the body, which is related to the protective effect of fermented black barley on the reproductive system of smoking mice. The previous study only shows that fermented black barley can significantly reduce the pulmonary congestion of mice caused by oil fume and improve the intestinal flora disorder, but it cannot be inferred that it can reduce the damage of reproductive organs and metabolic abnormalities of mice caused by smoking, because the gas composition of oil fume and tobacco smoke is completely different, and the action pathway and target molecule in the body are also different. For example, the regulation effect of fermented black barley on oxidative stress of lung tissue of smoking mice in the present application is different from that in the previous study, so it cannot be inferred that it can definitely play a regulatory role on the reproductive system and metabolic disorder of the body.

[0084] Example 2

[0085] On the basis of Example 1, the fermented black barley obtained by fermentation of Lactobacillus is further made into Lactobacillus fermented black barley food, Lactobacillus fermented black barley drink, and solid powder functional food obtained by freeze-drying or spray-drying processing of Lactobacillus fermented black barley.

[0086] The above describes in detail the preferred embodiments of the present application. It should be understood that those skilled in the art can make many modifications and changes without creative labor according to the concept of the present application. Therefore, any technical solution obtained by logical analysis, reasoning or limited experiment on the basis of the prior art according to the concept of the present application, including other fermented cereal foods obtained by similar fermentation technology, shall be within the protection scope determined by the claims.

Claims

1. The use of fermented grains in the preparation of a medicament for reducing tissue and organ damage caused by smoking, characterized in that, The organs and tissues include the heart and kidneys, and the raw material for the fermented grain is black barley; the fermentation strain used is Lactobacillus plantarum.

2. The use of a fermented grain in the preparation of a drug to reduce smoking-induced damage to the male reproductive system, wherein the raw material for the fermented grain is black barley; and the fermentation strain used is Lactobacillus plantarum.

3. The use as described in claim 1 or 2, characterized in that, The smoking includes active smoking and / or passive smoking.

4. The use as described in claim 1 or 2, characterized in that, The drug is a fermented grain liquid preparation, fermented grain powder, fermented grain capsule, fermented grain tablet, or fermented grain pill.

5. The use as described in claim 1 or 2, characterized in that, The fermented grain is prepared by a method comprising the following steps: S1. After crushing the grain, pass it through a 60-80 mesh sieve, add pure water at a mass ratio of 1:5 to 1:7, stir and heat to 63-67℃. S2. Add α-high-temperature amylase at a rate of 9.5-10.5 U / g, and hydrolyze for 35-45 minutes. After hydrolysis, obtain whole grain culture medium. S3. The grain culture medium is sterilized by high pressure, and after cooling, the activated fermentation inoculum is added at an inoculation rate of 2-5%. Fermentation is carried out at 25-35℃ for 24-40 hours on a shaker at a speed of 90-110 rpm.

6. The use according to claim 5, characterized in that, In step S3, the activated fermentation culture solution contains 10 live bacteria. 9 CFU / ml; viable cell count at the end of fermentation: 10 8 CFU / ml.

Images