WO2022151790A1 - 一种干预慢性肾炎及尿毒症的长双歧杆菌膳食组合及应用 - Google Patents

一种干预慢性肾炎及尿毒症的长双歧杆菌膳食组合及应用 Download PDF

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WO2022151790A1
WO2022151790A1 PCT/CN2021/125679 CN2021125679W WO2022151790A1 WO 2022151790 A1 WO2022151790 A1 WO 2022151790A1 CN 2021125679 W CN2021125679 W CN 2021125679W WO 2022151790 A1 WO2022151790 A1 WO 2022151790A1
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bifidobacterium longum
powder
intervening
chronic nephritis
uremia
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PCT/CN2021/125679
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French (fr)
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宋锦安
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润盈生物工程（上海）有限公司
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
    • A23C9/00—Milk preparations; Milk powder or milk powder preparations
    • A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
    • A23C9/123—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt
    • A23C9/1234—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt characterised by using a Lactobacillus sp. other than Lactobacillus Bulgaricus, including Bificlobacterium sp.
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L7/00—Cereal-derived products; Malt products; Preparation or treatment thereof
    • A23L7/10—Cereal-derived products
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23P—SHAPING OR WORKING OF FOODSTUFFS, NOT FULLY COVERED BY A SINGLE OTHER SUBCLASS
    • A23P10/00—Shaping or working of foodstuffs characterised by the products
    • A23P10/20—Agglomerating; Granulating; Tabletting
    • A23P10/28—Tabletting; Making food bars by compression of a dry powdered mixture
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/745—Bifidobacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/04—Preserving or maintaining viable microorganisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2400/00—Lactic or propionic acid bacteria
    • A23V2400/51—Bifidobacterium
    • A23V2400/533—Longum

Definitions

  • the invention relates to the field of health food, in particular to a dietary combination and application of Bifidobacterium longum for intervening chronic nephritis and uremia.
  • Chronic nephritis promotes the accumulation of organic residues in the body, which lead to a range of negative body effects, including increased inflammatory state, oxidative stress, cardiovascular dysfunction, and risk of death.
  • interventions are generally performed to limit the intake of certain food groups, such as fruits, vegetables and grains, in individuals with chronic nephritis. Restriction of intake of these foods often affects supplementation of micronutrients, dietary fiber, and phytochemicals with antioxidant and anti-inflammatory activity.
  • probiotic preparations can improve the host's antioxidant and anti-inflammatory activities, and can facilitate the absorption of nutrients by the host, but there is still a lack of technology and application of probiotic-related preparations to interfere with chronic nephritis and uremia.
  • the invention breaks through the difficulties of the prior art, and designs a dietary combination and application of Bifidobacterium longum for intervening chronic nephritis and uremia.
  • the present invention designs a dietary combination and application of Bifidobacterium longum intervening in chronic nephritis and uremia, including 100 mL of Bifidobacterium longum BL-G301 probiotic milk and 40 g of grain powder for tableting.
  • the preparation method of Bifidobacterium longum BL-G301 probiotic milk is as follows:
  • Step 1 Take out the Bifidobacterium longum BL-G301 probiotic bacteria powder frozen at -80°C and take it out for use.
  • the number of viable bacteria in the Bifidobacterium longum BL-G301 probiotic bacteria powder is ⁇ 1 ⁇ 10 10 CFU/ g;
  • Step 2 After the temperature reaches room temperature, the probiotic bacteria powder is added to the pasteurized milk until the number of viable bacteria is 7.4 ⁇ 10 8 ⁇ 5.4 ⁇ 10 8 CFU/100mL.
  • Cereal flour tablet contains 10% sugar and 0.5% sodium chloride.
  • the preparation method of Bifidobacterium longum BL-G301 probiotic bacteria powder is as follows:
  • Step 1 Inoculate the Bifidobacterium longum strains frozen at -80°C in the basal medium, and cultivate at a temperature of 37°C for 12 hours to obtain an activated seed medium;
  • Step 2 inoculate the activated seed medium in the fermentation medium with an inoculum amount of 2% to 5%, and cultivate at a temperature of 37° C. for 8 to 10 hours to obtain a fermented bacterial liquid;
  • Step 3 Collect the bacterial liquid, and centrifuge it with a refrigerated centrifuge.
  • the centrifugation conditions are: temperature 4 °C, rotation speed 6000 r/min, time 10 min, after centrifugation, the bacterial slurry and protective agent are emulsified in a ratio of 1:2, and vacuumed. Freeze-drying for 12h, the temperature of the cold trap of the freeze-drying machine is -80°C, to obtain bacterial powder;
  • Step 4 After the solid medium is heated and melted completely, cooled to about 50°C, take 0.5 mL of the appropriate dilution gradient dilution solution into a sterilized petri dish, pour the BMD medium and mix well, take 3 parallel plates for each gradient The total number of colonies was counted after anaerobic culture at 37°C for 48h.
  • the basal medium components in step 1 are: lactose 20g/L, peptone 10g/L, beef extract powder 10g/L, yeast extract 5g/L, L-cysteine 1g/L, dipotassium hydrogen phosphate 2g/L , Diamine hydrogen citrate 2g/L, sodium acetate 5g/L, magnesium sulfate heptahydrate 0.25g/L, manganese sulfate monohydrate 0.05g/L, Tween-80 1g/L.
  • Fermentation medium components in step 2 glucose 20g/L, peptone 10g/L, beef extract powder 10g/L, yeast extract 5g/L, dipotassium hydrogen phosphate 2g/L, diamine hydrogen citrate 2g/L, acetic acid Sodium 5g/L, Magnesium Sulfate Heptahydrate 4.92g/L, L-cysteine 1g/L, Tryptophan 0.4g/L, Aspartic Acid 0.4g/L, Manganese Sulfate Monohydrate 0.05g/L, Tween-80 1g/L.
  • the protective agent components in step 3 are: skimmed milk powder 110 g/L, sucrose 40 g/L, and monosodium glutamate 20 g/L.
  • composition of BMD medium in step 4 lactose 5g/L, tryptone 10g/L, beef extract powder 4g/L, yeast extract 3g/L, sodium chloride 4g/L, cysteine hydrochloride 1g/L , Mupirocin lithium salt 25mg/L, Tween-80 1g/L; 0.8g/L agar powder.
  • the classification name of Bifidobacterium longum BL-G301 strain is Bifidobacterium longum BL-G301 (Bifidobacterium longum BL-G301).
  • the preservation number is CCTCC NO: M2013689, and the preservation time is: December 23, 2013.
  • the preparation method of Bifidobacterium longum BL-G301 probiotic milk is used as a dietary combination together with the grain powder tablet to improve the oxidative stress in the human intestinal tract and reduce the levels of inflammatory factors and urinary toxins in the human body.
  • the present invention uses probiotics to intervene in chronic nephritis, which can reduce the level of oxidative stress in patients with chronic nephritis; improve the level of inflammatory factors and urinary toxins in patients with chronic nephritis;
  • the intestinal flora is healthier, the beneficial bacteria are increased, the harmful bacteria are reduced, and the flora structure is more conducive to intestinal health.
  • Fig. 1 is a bar graph showing the concentration of oxidative stress markers in the blood of patients after the intervention of the present invention.
  • Figure 2 is a bar graph showing the concentration of inflammatory markers in the blood of patients after the intervention of the present invention.
  • Figure 3 is a bar graph showing the concentration of uremic toxins in the blood of patients after the intervention of the present invention.
  • Fig. 4 is a bar graph of pH value and concentration of organic acids in stool samples of patients after the intervention of the present invention.
  • the classification name of the Bifidobacterium longum BL-G301 strain used in the present invention is Bifidobacterium longum BL-G301 (Bifidobacterium longum BL-G301). : Wuhan University, Wuhan, China, preservation number CCTCC NO: M2013689, preservation time: December 23, 2013.
  • the invention designs a Bifidobacterium longum dietary combination for intervening chronic nephritis and uremia, including 100 mL of Bifidobacterium longum BL-G301 probiotic milk and 40 g of grain powder tableting.
  • the preparation method of Bifidobacterium longum BL-G301 probiotic milk is as follows:
  • Step 1 Take out the Bifidobacterium longum BL-G301 probiotic bacteria powder frozen at -80°C and take it out for use.
  • the number of viable bacteria in the Bifidobacterium longum BL-G301 probiotic bacteria powder is ⁇ 1 ⁇ 10 10 CFU/ g;
  • Step 2 After the temperature reaches room temperature, the probiotic bacteria powder is added to the pasteurized milk until the number of viable bacteria is 7.4 ⁇ 10 8 ⁇ 5.4 ⁇ 10 8 CFU/100mL.
  • Cereal flour tablet contains 10% sugar and 0.5% sodium chloride.
  • the preparation method of Bifidobacterium longum BL-G301 probiotic bacteria powder is as follows:
  • Step 1 Inoculate the Bifidobacterium longum strains frozen at -80°C in the basal medium, and cultivate at a temperature of 37°C for 12 hours to obtain an activated seed medium;
  • Step 2 inoculate the activated seed medium in the fermentation medium with an inoculum amount of 2% to 5%, and cultivate at a temperature of 37° C. for 8 to 10 hours to obtain a fermented bacterial liquid;
  • Step 3 Collect the bacterial liquid and centrifuge with a refrigerated centrifuge.
  • the centrifugation conditions are: temperature 4 °C, rotating speed 6000 r/min, time 10 min, after centrifugation, emulsification of bacterial slurry and protective agent in a ratio of 1:2, and vacuum Freeze-drying for 12h, the temperature of the cold trap of the freeze-drying machine is -80°C, to obtain bacterial powder;
  • Step 4 After the solid medium is heated and melted completely, cooled to about 50°C, take 0.5 mL of the appropriate dilution gradient dilution solution into a sterilized petri dish, pour the BMD medium and mix well, take 3 parallel plates for each gradient The total number of colonies was counted after anaerobic culture at 37°C for 48h.
  • the basal medium components in step 1 are: lactose 20g/L, peptone 10g/L, beef extract powder 10g/L, yeast extract 5g/L, L-cysteine 1g/L, dipotassium hydrogen phosphate 2g/L , Diamine hydrogen citrate 2g/L, sodium acetate 5g/L, magnesium sulfate heptahydrate 0.25g/L, manganese sulfate monohydrate 0.05g/L, Tween-80 1g/L.
  • Fermentation medium components in step 2 glucose 20g/L, peptone 10g/L, beef extract powder 10g/L, yeast extract 5g/L, dipotassium hydrogen phosphate 2g/L, diamine hydrogen citrate 2g/L, acetic acid Sodium 5g/L, Magnesium Sulfate Heptahydrate 4.92g/L, L-cysteine 1g/L, Tryptophan 0.4g/L, Aspartic Acid 0.4g/L, Manganese Sulfate Monohydrate 0.05g/L, Tween-80 1g/L.
  • the protective agent components in step 3 are: skimmed milk powder 110 g/L, sucrose 40 g/L, and monosodium glutamate 20 g/L.
  • composition of BMD medium in step 4 lactose 5g/L, tryptone 10g/L, beef extract powder 4g/L, yeast extract 3g/L, sodium chloride 4g/L, cysteine hydrochloride 1g/L , Mupirocin lithium salt 25mg/L, Tween-80 1g/L; 0.8g/L agar powder.
  • Bifidobacterium longum BL-G301 probiotic milk and grain powder tablet are used as a dietary combination to improve the oxidative stress in the human intestine and reduce the level of inflammatory factors and urinary toxins in the human body.
  • the prepared Bifidobacterium longum BL-G301 probiotic milk and sorghum flour extruded tablets were used as the experimental group, and the daily consumption of 100 mL of probiotic milk was matched with 40 g of sorghum flour extruded tablets; the control group was the bar without probiotics.
  • the daily dosage is 100 mL of probiotic milk and 40 g of corn flour extruded tablets.
  • Blood samples were collected by qualified professionals before hemodialysis, centrifuged, and immediately stored at -80°C.
  • SOD superoxide dismutase
  • MDA malondialdehyde
  • TAC total antioxidant capacity
  • the prepared Bifidobacterium longum BL-G301 probiotic milk and sorghum flour extruded tablets were used as the dietary combination of the experimental group, and the daily consumption of 100 mL of probiotic milk was matched with 40 g of sorghum flour extruded tablets; the control group did not contain probiotics.
  • of pasteurized milk and cornmeal extruded tablets use 100mL of probiotic milk with 40g of cornmeal extruded tablets per day.
  • Blood samples were collected by qualified professionals before hemodialysis, centrifuged, and immediately stored at -80°C.
  • IL-6 concentrations of IL-6, IL-10, TNF- ⁇ and C-reactive protein (CRP) that can reflect the concentration of inflammatory factors in the samples were detected.
  • CRP C-reactive protein
  • the prepared Bifidobacterium longum BL-G301 probiotic milk and sorghum flour extruded tablets were used as the dietary combination of the experimental group, and the daily consumption of 100 mL of probiotic milk was matched with 40 g of sorghum flour extruded tablets; the control group did not contain probiotics.
  • of pasteurized milk and cornmeal extruded tablets use 100mL of probiotic milk with 40g of cornmeal extruded tablets per day.
  • Blood samples were collected by qualified professionals before hemodialysis, centrifuged, and immediately stored at -80°C.
  • the prepared Bifidobacterium longum BL-G301 probiotic milk and sorghum flour extruded tablets were used as the dietary combination of the experimental group, and the daily consumption of 100 mL of probiotic milk was matched with 40 g of sorghum flour extruded tablets; the control group did not contain probiotics.
  • of pasteurized milk and cornmeal extruded tablets use 100mL of probiotic milk with 40g of cornmeal extruded tablets per day.
  • Fecal samples were collected by volunteers in sterile vials, and the collected fecal samples were snap-frozen and stored at -18°.
  • Test stool samples for pH and organic acid concentrations such as acetic, propionic, and butyric acids.

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Abstract

一种干预慢性肾炎及尿毒症的长双歧杆菌膳食组合及应用，包括100mL长双歧杆菌BL-G301益生菌牛乳和40g谷物粉压片，制备方法为将-80℃冷冻保藏的益生菌菌粉取出，温度至室温后，将益生菌菌粉加至巴氏灭菌牛乳中；将长双歧杆菌BL-G301益生菌牛乳的制备方法制备获得物和谷物粉压片一同作为膳食搭配，改善人体肠道内的氧化应激，降低人体内炎症因子和尿毒素水平。

Description

一种干预慢性肾炎及尿毒症的长双歧杆菌膳食组合及应用 技术领域
本发明涉及保健食品领域，具体的讲是一种干预慢性肾炎及尿毒症的长双歧杆菌膳食组合及应用。
背景技术
基于发病率和患病率，慢性肾炎已成为世界范围内的一个主要公共卫生问题，在过去4年中，慢性肾炎患病率增长了约5％，该疾病的总体平均流行率达到了13.4％。
慢性肾炎会促进体内有机残留物的积累，这些残留物会带来一系列机体负面作用，包括加重炎症状态、氧化应激、心血管功能障碍和死亡风险。目前尚无较好的慢性肾炎药物控制方法，为了控制这种积累，一般是对患有慢性肾炎的个体限制某些食物群的摄入，如水果、蔬菜和谷物，进行干预。而对这些食物的摄入限制往往会影响微量营养素、膳食纤维和具有抗氧化和抗炎活性的植物化学物质的补充。现在研究表明益生菌制剂的补充可以改善宿主抗氧化和抗炎活性，并能利于宿主营养的吸收，但是目前尚缺乏用益生菌相关制剂干预慢性肾炎及尿毒症的技术和应用。
因此设计一种干预慢性肾炎及尿毒症的长双歧杆菌膳食组合及应用是十分有必要的。
发明内容
本发明突破了现有技术的难题，设计了一种干预慢性肾炎及尿毒症的长双歧杆菌膳食组合及应用。
为了达到上述目的，本发明设计了一种干预慢性肾炎及尿毒症的长双歧杆菌膳食组合及应用，包括100mL长双歧杆菌BL-G301益生菌牛乳和40g谷物粉压片。
长双歧杆菌BL-G301益生菌牛乳的制备方法如下：
步骤1：将-80℃冷冻保藏的长双歧杆菌BL-G301益生菌菌粉，取出备用，所述的长双歧杆菌BL-G301益生菌粉中的活菌数≥1×10 10CFU/g；
步骤2：温度至室温后，将益生菌菌粉加至巴氏灭菌牛乳中，至活菌数为7.4×10 8±5.4×10 8CFU/100mL。
谷物粉压片包括10％白糖和0.5％的氯化钠。
长双歧杆菌BL-G301益生菌菌粉的制备方法如下：
步骤1：将-80℃冷冻保藏的长双歧杆菌菌株接种在基础培养基中，并在37℃的温度下培养12h，得到活化的种子培养基；
步骤2：将活化的种子培养基以2％～5％的接种量分别接种在发酵培养基中，在37℃的温度下培养8～10h，得到发酵好的菌液；
步骤3：收集菌液，采用冷冻离心机进行离心，离心条件为：温度4℃，转速6000r/min，时间10min，离心后将菌泥和保护剂，按照1：2的比例进行乳化，并真空冷冻干燥12h，冻干机的冷阱温度为-80℃，获得菌粉；
步骤4：将固体培养基加热完全融化后，冷却至50℃左右，取合适的稀释梯度的稀释液0.5mL于灭菌培养皿中，倾注BMD培养基后混匀，每个梯度取3个平行样，37℃厌氧培养48h后统计菌落总数。
步骤1中的基础培养基成分为：乳糖20g/L、蛋白胨10g/L、牛肉浸粉10g/L、酵母膏5g/L、L-半胱氨酸1g/L、磷酸氢二钾2g/L、柠檬酸氢二胺2g/L、乙酸钠5g/L、七水硫酸镁0.25g/L、一水硫酸锰0.05g/L、吐温－80 1g/L。
步骤2中的发酵培养基成分：葡萄糖20g/L、蛋白胨10g/L、牛肉浸粉10g/L、酵母膏5g/L、磷酸氢二钾2g/L、柠檬酸氢二胺2g/L、乙酸钠5g/L、七水硫酸镁4.92g/L、L-半胱氨酸1g/L、色氨酸0.4g/L，天冬氨酸0.4g/L，一水硫酸锰0.05g/L、吐温-80 1g/L。
步骤3中的保护剂成分为：脱脂奶粉110g/L、蔗糖40g/L、味精20g/L。
步骤4中的BMD培养基组成：乳糖5g/L、胰蛋白胨10g/L、牛肉浸粉4g/L、酵母膏3g/L、氯化钠4g/L、半胱氨酸盐酸盐1g/L、莫匹罗星锂盐25mg/L、吐温－80 1g/L；0.8g/L琼脂粉。
长双歧杆菌BL-G301菌株的分类名为长双歧杆菌BL-G301(Bifidobacterium longum BL-G301)，保藏单位为：中国典型培养物保藏中心(CCTCC)，保藏单位地址为：中国武汉武汉大学，保藏编号为CCTCC NO：M2013689，保藏时间为：2013年12月23日。
将长双歧杆菌BL-G301益生菌牛乳的制备方法制备获得物和谷物粉压片一同作为膳食搭配，改善人体肠道内的氧化应激，降低人体内炎症因子和尿毒素水平。
本发明与现有技术相比，采用益生菌干预慢性肾炎，可以降低慢性肾炎患者氧化应激水平；改善慢性肾炎患者炎症因子水平和尿毒素水平；改善粪便pH值，提升粪便有机酸含量，肠道菌群更加健康，有益菌增多，有害菌减少，菌群结构更利于肠道健康。
附图说明
图1为本发明干预后患者血液中氧化应激标志物的浓度柱状图。
图2为本发明干预后患者血液中的炎症标志物的浓度柱状图。
图3为本发明干预后患者血液中尿毒症毒素的浓度柱状图。
图4为本发明干预后患者粪便样本的pH值和有机酸的浓度柱状图。
具体实施方式
现对本发明做进一步描述。
本发明中采用的长双歧杆菌BL-G301菌株的分类名为长双歧杆菌BL-G301(Bifidobacterium longum BL-G301)，保藏单位为：中国典型培养物保藏中心(CCTCC)，保藏单位地址为：中国武汉武汉大学，保藏编号为CCTCC NO：M2013689，保藏时间为：2013年12月23日。
本发明设计了一种干预慢性肾炎及尿毒症的长双歧杆菌膳食组合，包括100mL长双歧杆菌BL-G301益生菌牛乳和40g谷物粉压片。
长双歧杆菌BL-G301益生菌牛乳的制备方法如下：
步骤1：将-80℃冷冻保藏的长双歧杆菌BL-G301益生菌菌粉，取出备用，所述的长双歧杆菌BL-G301益生菌粉中的活菌数≥1×10 10CFU/g；
步骤2：温度至室温后，将益生菌菌粉加至巴氏灭菌牛乳中，至活菌数为7.4×10 8±5.4×10 8CFU/100mL。
谷物粉压片包括10％白糖和0.5％的氯化钠。
长双歧杆菌BL-G301益生菌菌粉的制备方法如下：
步骤1：将-80℃冷冻保藏的长双歧杆菌菌株接种在基础培养基中，并在37℃的温度下培养12h，得到活化的种子培养基；
步骤2：将活化的种子培养基以2％～5％的接种量分别接种在发酵培养基中，在37℃的温度下培养8～10h，得到发酵好的菌液；
步骤3：收集菌液，采用冷冻离心机进行离心，离心条件为：温度4℃，转速6000r/min，时间10min，离心后将菌泥和保护剂，按照1：2的比例进行乳化，并真空冷冻干燥12h，冻干机的冷阱温度为-80℃，获得菌粉；
步骤4：将固体培养基加热完全融化后，冷却至50℃左右，取合适的稀释梯度的稀释液0.5mL于灭菌培养皿中，倾注BMD培养基后混匀，每个梯度取3个平行样，37℃厌氧培养48h后统计菌落总数。
步骤1中的基础培养基成分为：乳糖20g/L、蛋白胨10g/L、牛肉浸粉10g/L、酵母膏5g/L、L-半胱氨酸1g/L、磷酸氢二钾2g/L、柠檬酸氢二胺2g/L、乙酸钠5g/L、七水硫酸镁0.25g/L、一水硫酸锰0.05g/L、吐温－80 1g/L。
步骤2中的发酵培养基成分：葡萄糖20g/L、蛋白胨10g/L、牛肉浸粉10g/L、酵母膏5g/L、磷酸氢二钾2g/L、柠檬酸氢二胺2g/L、乙酸钠5g/L、七水硫酸镁4.92g/L、L-半胱氨酸1g/L、色氨酸0.4g/L，天冬氨酸0.4g/L，一水硫酸锰0.05g/L、吐温-80 1g/L。
步骤3中的保护剂成分为：脱脂奶粉110g/L、蔗糖40g/L、味精20g/L。
步骤4中的BMD培养基组成：乳糖5g/L、胰蛋白胨10g/L、牛肉浸粉4g/L、酵母膏3g/L、氯化钠4g/L、半胱氨酸盐酸盐1g/L、莫匹罗星锂盐25mg/L、吐温－80 1g/L；0.8g/L琼脂粉。
将长双歧杆菌BL-G301益生菌牛乳和谷物粉压片一同作为膳食搭配，改善人体肠道内的氧化应激，降低人体内炎症因子和尿毒素水平。
结合实例对本发明的具体实施方式做进一步说明。
实施例1：
将制备好的长双歧杆菌BL-G301益生菌牛乳和高粱粉挤压片作为实验组，每天使用量为100mL益生菌的牛乳搭配40g高粱粉挤压片；对照组为不含益生菌的巴氏灭菌牛乳和玉米粉挤压片，每天使用量为100mL益生菌的牛乳搭配40g玉米粉挤压片。
选取58个患有慢性肾炎的志愿者，参与者年龄超过18岁，每周进行三至四次血液透析，至少三个月。人员随机分为两组，实验组和对照组各29人。
血液样本的采集是在血液透析前由合格的专业人员收集的后，将血液样本离心，立即储存在-80℃的状态。
检测样本中超氧化物歧化酶的酶活性(SOD)、丙二醛(MDA)和总抗氧化能力(TAC)。
结合图1，在氧化应激方面，得到益生菌BL-G301的实验组(SG)干预后血清MDA下降(p＜0.05)，SOD和TAC与对照组(CG)相比增加(p＜0.05)。
实施例2：
将制备好的长双歧杆菌BL-G301益生菌牛乳和高粱粉挤压片作为实验组膳食搭配，每天使用量为100mL益生菌的牛乳搭配40g高粱粉挤压片；对照组为不含益生菌的巴氏灭菌牛乳和玉米粉挤压片，每天使用量为100mL益生菌的牛乳搭配40g玉米粉挤压片。
选取58个患有慢性肾炎的志愿者，参与者年龄超过18岁，每周进行三至四次血液透析，至少三个月。人员随机分为两组，实验组和对照组各29人。
血液样本的采集是在血液透析前由合格的专业人员收集的后，将血液样本离心，立即储存在-80℃的状态。
检测样本中能反应炎症因子浓度的IL-6、IL-10和TNF-α浓度和C反应蛋白浓度(CRP)。
结合图2，与对照组(CG)相比，实验组(SG)CRP浓度降低(p＜0.05)；而IL-6、IL-10和TNF-α细胞因子间无明显变化(p＞0.05)。
实施例3：
将制备好的长双歧杆菌BL-G301益生菌牛乳和高粱粉挤压片作为实验组膳食搭配，每天使用量为100mL益生菌的牛乳搭配40g高粱粉挤压片；对照组为不含益生菌的巴氏灭菌牛乳和玉米粉挤压片，每天使用量为100mL益生菌的牛乳搭配40g玉米粉挤压片。
选取58个患有慢性肾炎的志愿者，参与者年龄超过18岁，每周进行三至四次血液透析，至少三个月。人员随机分为两组，实验组和对照组各29人。
血液样本的采集是在血液透析前由合格的专业人员收集的后，将血液样本离心，立即储存在-80℃的状态。
检测样本中代表尿毒症程度的三个指标：血清硫酸吲哚、硫酸对甲酚和吲哚3-乙酸。
结合图3，与对照组(CG)相比，实验组(SG)血清硫酸吲哚和硫酸对甲酚浓度更低(p＜0.05)；而吲哚3-乙酸的浓度无明显变化(p＞0.05)。
实施例4
将制备好的长双歧杆菌BL-G301益生菌牛乳和高粱粉挤压片作为实验组膳食搭配，每天使用量为100mL益生菌的牛乳搭配40g高粱粉挤压片；对照组为不含益生菌的巴氏灭菌牛乳和玉米粉挤压片，每天使用量为100mL益生菌的牛乳搭配40g玉米粉挤压片。
选取58个患有慢性肾炎的志愿者，参与者年龄超过18岁，每周进行三至四次血液透析，至少三个月。人员随机分为两组，实验组和对照组各29人。
粪便样本由志愿者采用无菌瓶收集，收集到的粪便样本快速冷冻保存在-18°。
检测粪便样本的pH值和乙酸，丙酸和丁酸等有机酸浓度。
结合图4，与对照组(CG)相比，实验组(SG)的终点粪便pH值更低(p＜0.05)，实验组(SG)的终点乙酸，丙酸和丁酸有机酸浓度更高。
结合图1～图4表明长双歧杆菌BL-G301益生菌牛乳的制备方法制备获得物和谷物粉压片一同作为膳食搭配可以降低慢性肾炎患者血液尿毒症毒素浓度的水平，同时可以降低慢性肾炎患者粪便pH值和提升粪便有机酸浓度。

Claims (10)

  1. 一种干预慢性肾炎及尿毒症的长双歧杆菌膳食组合，其特征在于：包括100mL长双歧杆菌BL-G301益生菌牛乳和40g谷物粉压片。
  2. 根据权利要求1所述的一种干预慢性肾炎及尿毒症的长双歧杆菌膳食组合，其特征在于：所述长双歧杆菌BL-G301益生菌牛乳的制备方法如下：
    步骤1：将-80℃冷冻保藏的长双歧杆菌BL-G301益生菌菌粉，取出备用，所述的长双歧杆菌BL-G301益生菌粉中的活菌数≥1×10 10CFU/g；
    步骤2：温度至室温后，将益生菌菌粉加至巴氏灭菌牛乳中，至活菌数为7.4×10 8±5.4×10 8CFU/100mL。
  3. 根据权利要求1所述的一种干预慢性肾炎及尿毒症的长双歧杆菌膳食组合，其特征在于：所述的谷物粉压片包括10％白糖和0.5％的氯化钠。
  4. 根据权利要求2所述的一种干预慢性肾炎及尿毒症的长双歧杆菌膳食组合，其特征在于：所述的长双歧杆菌BL-G301益生菌菌粉的制备方法如下：
    步骤1：将-80℃冷冻保藏的长双歧杆菌菌株接种在基础培养基中，并在37℃的温度下培养12h，得到活化的种子培养基；
    步骤2：将活化的种子培养基以2％～5％的接种量分别接种在发酵培养基中，在37℃的温度下培养8～10h，得到发酵好的菌液；
    步骤3：收集菌液，采用冷冻离心机进行离心，离心条件为：温度4℃，转速6000r/min，时间10min，离心后将菌泥和保护剂，按照1：2的比例进行乳化，并真空冷冻干燥12h，冻干机的冷阱温度为-80℃，获 得菌粉；
    步骤4：将固体培养基加热完全融化后，冷却至50℃左右，取合适的稀释梯度的稀释液0.5mL于灭菌培养皿中，倾注BMD培养基后混匀，每个梯度取3个平行样，37℃厌氧培养48h后统计菌落总数。
  5. 根据权利要求4所述的一种干预慢性肾炎及尿毒症的长双歧杆菌膳食组合，其特征在于：所述步骤1中的基础培养基成分为：乳糖20g/L、蛋白胨10g/L、牛肉浸粉10g/L、酵母膏5g/L、L-半胱氨酸1g/L、磷酸氢二钾2g/L、柠檬酸氢二胺2g/L、乙酸钠5g/L、七水硫酸镁0.25g/L、一水硫酸锰0.05g/L、吐温－80 1g/L。
  6. 根据权利要求4所述的一种干预慢性肾炎及尿毒症的长双歧杆菌膳食组合，其特征在于：所述步骤2中的发酵培养基成分：葡萄糖20g/L、蛋白胨10g/L、牛肉浸粉10g/L、酵母膏5g/L、磷酸氢二钾2g/L、柠檬酸氢二胺2g/L、乙酸钠5g/L、七水硫酸镁4.92g/L、L-半胱氨酸1g/L、色氨酸0.4g/L，天冬氨酸0.4g/L，一水硫酸锰0.05g/L、吐温-80 1g/L。
  7. 根据权利要求4所述的一种干预慢性肾炎及尿毒症的长双歧杆菌膳食组合，其特征在于：所述步骤3中的保护剂成分为：脱脂奶粉110g/L、蔗糖40g/L、味精20g/L。
  8. 根据权利要求4所述的一种干预慢性肾炎及尿毒症的长双歧杆菌膳食组合，其特征在于：所述步骤4中的BMD培养基组成：乳糖5g/L、胰蛋白胨10g/L、牛肉浸粉4g/L、酵母膏3g/L、氯化钠4g/L、半胱氨酸盐酸盐1g/L、莫匹罗星锂盐25mg/L、吐温－80 1g/L；0.8g/L琼 脂粉。
  9. 根据权利要求1所述的一种干预慢性肾炎及尿毒症的长双歧杆菌膳食组合，其特征在于：所述的长双歧杆菌BL-G301菌株的分类名为长双歧杆菌BL-G301(Bifidobacterium longum BL-G301)，保藏单位为：中国典型培养物保藏中心(CCTCC)，保藏单位地址为：中国武汉武汉大学，保藏编号为CCTCC NO：M2013689，保藏时间为：2013年12月23日。
  10. 一种干预慢性肾炎及尿毒症的长双歧杆菌膳食组合的应用，其特征在于：将如权利要求1～8所述的长双歧杆菌BL-G301益生菌牛乳的制备方法制备获得物和谷物粉压片一同作为膳食搭配，改善人体肠道内的氧化应激，降低人体内炎症因子和尿毒素水平。
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CN112007052A (zh) * 2020-09-22 2020-12-01 上海泓商生物科技有限公司 护肾益生菌组合物
CN112715660A (zh) * 2021-01-18 2021-04-30 润盈生物工程（上海）有限公司 一种干预慢性肾炎及尿毒症的长双歧杆菌膳食组合及应用

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CN116585360A (zh) * 2023-05-24 2023-08-15 微康益生菌(苏州)股份有限公司 一种改善慢性肾病的益生菌剂及其应用
CN116585360B (zh) * 2023-05-24 2023-11-14 微康益生菌(苏州)股份有限公司 一种改善慢性肾病的益生菌剂及其应用

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