CN113234612A - Lactobacillus fermentum ZS40 having preventive effect on colitis - Google Patents
Lactobacillus fermentum ZS40 having preventive effect on colitis Download PDFInfo
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- CN113234612A CN113234612A CN202110160207.5A CN202110160207A CN113234612A CN 113234612 A CN113234612 A CN 113234612A CN 202110160207 A CN202110160207 A CN 202110160207A CN 113234612 A CN113234612 A CN 113234612A
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- lactobacillus fermentum
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- staphylococcus xylosus
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Abstract
The invention discloses Lactobacillus fermentum ZS40 with a prevention effect on colitis, wherein the preservation number of the Lactobacillus fermentum ZS40 is CGMCC NO. 18226. The strain has stronger gastric acid resistance, and the survival rate of the strain after being treated by artificial gastric juice with the pH value of 3.0 for 3 hours reaches 79.32 percent; the growth efficiency of the bacterium in 0.30% of bile salt reaches 15.31% of that in the bile salt-free culture. Researches show that the strain can inhibit the phenomena of colon shortening, colon injury, intestinal wall thickening and the like of mice, can also increase the activities of T-SOD and CAT, reduce the contents of MPO and MDA, regulate the imbalance of proinflammatory cytokines and anti-inflammatory cytokines, and better relieve inflammation by inhibiting the activation of NF-kB and MAPK signal pathways. In conclusion, the strain can effectively relieve the symptoms of the colitis mouse and can be used for preventing and treating colitis.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to Lactobacillus fermentum ZS40 with a prevention effect on colitis.
Background
Colitis is a chronic nonspecific inflammatory bowel disease, and the main symptoms include abdominal pain, diarrhea, hematochezia, and emaciation. The disease condition is mild and severe, and the disease is frequently recurrent, which seriously affects the life quality of patients and may further worsen to colon cancer. Colitis occurs around the world, but the incidence rates of people of different ethnic groups, regions, ages and sexes are different, and the pathogenesis of colitis is related to the multiple factors and the multiple-link combined action of immune dysfunction, intestinal flora imbalance, immune response imbalance and the like of patients. In recent years, epidemiological data have shown an increasing trend in the incidence and prevalence of colitis, possibly due to changes in people's lifestyle, diet and work and rest. Colitis has become one of the most common diseases of the digestive system in china. Common medicines for treating colitis include sulfasalazine, aminosalicylic acid, glucocorticoid and the like, but continuous taking of the medicines can generate certain side effects on a human body, so that the finding of a way with high safety and small toxic and side effects to relieve colitis has become a hot point of scientific research in recent years.
Xinjiang is located in the northwest of China and is one of the places where minority nationalities gather, and the traditional dietary culture makes dairy products an indispensable food in daily life. The traditional dairy product manufacturing process is more ecological and natural, the types of probiotics in the dairy product are richer, and various lactic acid bacteria resources are contained. The lactobacillus with the probiotic function is screened from the traditional dairy product, so that the lactobacillus seed bank can be enriched, and the beneficial bacterium raw material can be provided for industrial production.
Probiotics are a group of microorganisms which are present in the gastrointestinal tract of the human body and have activity beneficial to human health, and can exert various physiological and biochemical functions in the human body, such as: regulating intestinal flora balance, improving intestinal mucosa function, inhibiting pathogenic intestinal bacteria growth, and preventing gastrointestinal infection and inflammatory bowel disease. Lactic acid bacteria are considered the safest probiotic and many researchers have studied the efficacy of lactic acid bacteria, such as:
woo JY et al investigated the relief effect of plant-type lactobacillus pentosus C29 on d-galactose-induced memory impairment accelerated aging mice. The results indicate that treatment with Lactobacillus pentosus C29 can improve d-galactose-induced memory impairment (Woo JY, Wan G, Kim KA, et al, Lactobacillus pentosus var. plantarum C29 amino peptides memory impact and inflamaging in a d-galactose-induced accessed cultured mouse model [ J ]. Anaerobe,2014,27, 22-26.).
Long X et al investigated the effect of lactobacillus plantarum KFY04 on PPAR pathways in obese mice. The results show that Lactobacillus plantarum KFY04 can inhibit obesity, reduce oxidative damage and inflammation in mice (Long X, Zeng X, Tan F, et al, Lactobacillus plantarum KFY04 prevention organisms in micro tissue and inflammation [ J ]. Food & Function,2020, 11.).
TH et al, the mitigating effect of lactobacillus plantarum LP33 on liver damage in Pb-poisoned rats was studied. The results show that Lactobacillus plantarum LP33 can reduce the oxidative stress and inflammatory response of liver in rats induced by Pb and increase Pb excretion in rats with Pb poisoning (Hu T, Song JJ, Zeng WY et al Lactobacillus plantarum LP33 expressed proteins Pb-induced metabolic activity and inflammation and promoting Pb excretion [ J ]. Food and Chemical reactivity, 2020,143,111533).
Therefore, the use of probiotics for the prevention and treatment of colitis is probably a potential approach, and lactic acid bacterial strains with special efficacy are screened and used for production, which may make better use of their value.
Disclosure of Invention
The invention aims to separate and identify lactic acid bacteria in traditional fermented yak yogurt, and investigate the effect of the lactic acid bacteria on ulcerative colitis so as to enrich lactic acid bacteria strain resources.
Through research, the invention provides the following technical scheme:
the invention provides Lactobacillus fermentum ZS40 with a preservation number of CGMCC NO.18226 for preventing colitis.
According to the invention, lactic acid bacteria in traditional fermented yak yogurt are separated and identified, one of the Lactobacillus fermentum is named as ZS40, and is preserved in China general microbiological culture Collection center (CGMCC for short, address: No. 3 Xilu No.1 North Chen of the sunward area in Beijing city) in 2019, 15 months and 15 days, and the preservation number is CGMCC NO. 18226.
The Lactobacillus fermentum ZS40 has the morphological characteristics that: the characteristics of the thallus are as follows: the gram stain is bluish purple, the bacterial strain is gram positive, the cell morphology has long rods and short rods, and no budding reproduction exists;
colony characteristics: the colony color is white or milk white, the shape is round, the edge is neat, and the surface is moist and smooth.
The survival rate of the Lactobacillus fermentum ZS40 after being treated by artificial gastric juice with pH of 3.0 for 3 hours reaches 75-85 percent; the growth efficiency of the bacteria in 0.30 wt% of bile salt reaches 10-20 wt% of that of the bacteria cultured without bile salt. The results show that the Lactobacillus fermentum ZS40 of the present invention has gastric acid resistance.
Preferably, the Lactobacillus fermentum ZS40 has strong gastric acid resistance, and the survival rate of the Lactobacillus fermentum treated by the artificial gastric juice with the ph of 3.0 for 3 hours reaches 79.32%; the growth efficiency of the bacterium in 0.30 wt% of bile salt reaches 15.31% of that in the bile salt-free culture.
Through research on a 3 wt% DSS-induced colitis mouse model, the research finds that Lactobacillus fermentum ZS40 can inhibit the phenomena of colon shortening, colon injury, intestinal wall thickening and the like of mice, can also increase the activities of T-SOD and CAT, reduce the contents of MPO and MDA, regulate the imbalance of proinflammatory cytokines and anti-inflammatory cytokines, and better relieve inflammation through inhibiting the activation of NF-kB and MAPK signal pathways. The strain can effectively relieve the symptoms of the mice with the colitis, and can be used for preventing and treating the colitis.
Further, the invention provides a Lactobacillus fermentum (ZS 40) leavening agent.
The leavening agent is prepared by inoculating Lactobacillus fermentum ZS40 into MRS liquid for cultureCulturing in culture medium at 37 deg.C for 12-18h for activation to obtain Lactobacillus fermentum ZS40 containing Lactobacillus fermentum of 1 × 106-1×108cfu/mL activated culture; the Lactobacillus fermentumZS40 containing Lactobacillus fermentum is 1 × 106-1×108Inoculating the activated cfu/mL culture in MRS liquid culture medium at 2-4 vol%, culturing for 16-24h, centrifuging at 4 deg.C and 4000r/min for 10-15min, collecting precipitate, and washing with sterile water for 3 times to obtain starter.
The MRS liquid culture medium is as follows: casein peptone 10.0g, beef extract 10.0g, yeast powder 5.0g, glucose 5.0g, sodium acetate 5.0g, diammonium citrate 2.0g, Tween 801.0 g, and K2HPO4 2.0g，MgSO4.7H2O 0.2g，MnSO4.H2O 0.05g，CaCO320.0g, agar 15.0g, tap water 1.0L, pH6.8, sterilization conditions: 121 ℃ for 15 min.
Further, the invention provides application of Lactobacillus fermentum (ZS 40) in fermenting meat products.
Further, the fermented meat product is fermented sausage; the fermented sausage comprises the following preparation steps:
(1) mincing 100 plus 200 parts by weight of fresh pork without fascia and tendon, and stirring to prepare pork stuffing;
(2) adding auxiliary materials into the pork stuffing obtained in the step (1) and pickling at 4 ℃;
(3) inoculating 0.12-0.24 weight part of leaven into the pork stuffing obtained in the step (2), and uniformly stirring to prepare the sausage; (4) fermenting the sausage obtained in the step (3) at 25-35 ℃ and RH 80-98% for 1-3 days;
(5) fermenting the sausage in the step (4) at 12-15 deg.C and RH 72-90% for 11-16 days;
(6) vacuum packaging to obtain fermented sausage;
the leavening agent is a Lactobacillus fermentum ZS40 leavening agent; the preparation method of the Lactobacillus fermentum ZS40 leavening agent comprises the steps of inoculating Lactobacillus fermentum ZS40 into an MRS liquid culture medium, culturing at 37 ℃ for 12-18h, and activating to obtain the Lactobacillus fermentum containing leavening agentLactobacillus fermentum ZS40 is 1 × 106-1×108cfu/mL activated culture; the Lactobacillus fermentumZS40 containing Lactobacillus fermentum is 1 × 106-1×108Inoculating the cfu/mL activated culture in an MRS liquid culture medium according to 2-4 vol%, culturing for 16-24h, centrifuging at 4 ℃ for 10-15min at 4000r/min, collecting precipitates, washing with sterile water for 3 times, and centrifuging at 4000r/min for 15-30min to obtain Lactobacillus fermentum ZS40 strain; adding the Lactobacillus fermentum ZS40 strain into 100mL of aqueous solution containing 0.08g/mL of skim milk powder and 0.08g/mL of glucose, mixing uniformly, and freeze-drying to obtain the Lactobacillus fermentum ZS40 leavening agent.
The auxiliary materials comprise 2 to 4 weight parts of salt, 0.2 to 0.4 weight part of monosodium glutamate, 1 to 2 weight parts of glucose, 0.1 to 0.2 weight part of pepper powder, 0.1 to 0.2 weight part of ginger powder, 0.001 to 0.002 weight part of sodium nitrite and 0.01 to 0.02 weight part of sodium nitrite;
in order to further improve the quality of the fermented sausage, the inventor researches and discovers that the fermented sausage prepared by introducing Staphylococcus xylosus (Staphylococcus xylosus) ATCC29971 and Lactobacillus fermentum (Lactobacillus fermentum) ZS40 can effectively improve the quality and color of the fermented sausage and improve the food safety. The possible reasons for this are: staphylococcus xylosus (Staphylococcus xylosus) ATCC29971 can release lipase and protease, enhance flavor of meat products, generate nitrite reductase, and improve color of products; the Lactobacillus fermentum ZS40 has the characteristics of high acid production speed, salt resistance, bacteriostasis and the like, can shorten the fermentation period and improve the food safety. The fermented sausage prepared by mixing Staphylococcus xylosus (Staphylococcus xylosus) ATCC29971 and Lactobacillus fermentum (Lactobacillus fermentum) ZS40 remarkably improves the number of lactic acid bacteria at the initial stage of fermentation and improves the fermentation capacity. The nitrite content and the TBA-N value are obviously reduced, and the sausage spoilage probability is reduced. The good flavor not only can endow the sausage with good quality, but also can increase the purchasing desire of consumers. Meanwhile, the edible safety of the meat products also attracts wide attention. The method is particularly important for improving the content of good flavor substances of the sausage and controlling the content of harmful biogenic amine in the sausage.
Preferably, the leavening agent is Staphylococcus xylosus (Staphylococcus xylosus) ATCC29971 leavening agent and Lactobacillus fermentum (Lactobacillus fermentum) ZS40 leavening agent according to the mass ratio of (1-3): (1-3) mixing.
The leaven is Staphylococcus xylosus (Staphylococcus xylosus) ATCC29971 leaven; the preparation method of the Staphylococcus xylosus (Staphylococcus xylosus) ATCC29971 leavening agent comprises the steps of inoculating Staphylococcus xylosus (Staphylococcus xylosus) ATCC29971 into an MRS liquid culture medium, culturing at 37 ℃ for 12-18h, and activating to obtain Staphylococcus xylosus (Staphylococcus xylosus) ATCC29971 with the volume of 1 x 106-1×108cfu/mL activated culture; the content of Staphylococcus xylosus (Staphylococcus xylosus) ATCC29971 was 1X 106-1×108Inoculating the cfu/mL activated culture in MRS liquid culture medium at 2-4 vol%, culturing for 16-24h, centrifuging at 4 deg.C and 4000r/min for 10-15min, collecting precipitate, washing with sterile water for 3 times, centrifuging at 4000r/min for 15-30min to obtain Staphylococcus xylosus (Staphylococcus xylosus) ATCC29971 strain; adding the obtained Staphylococcus xylosus (Staphylococcus xylosus) ATCC29971 strain into 100mL of aqueous solution containing 0.08g/mL of skim milk powder and 0.08g/mL of glucose, uniformly mixing, and freeze-drying to obtain the Staphylococcus xylosus (Staphylococcus xylosus) ATCC29971 leavening agent.
The Lactobacillus fermentum ZS40 leavening agent is Lactobacillus fermentum ZS40 leavening agent; the preparation method of the leavening agent comprises the steps of inoculating Lactobacillus fermentum ZS40 into MRS liquid culture medium, culturing at 37 ℃ for 12-18h for activation, and obtaining Lactobacillus fermentum ZS40 containing Lactobacillus fermentum of 1 multiplied by 106-1×108cfu/mL activated culture; the Lactobacillus fermentumZS40 containing Lactobacillus fermentum is 1 × 106-1×108Inoculating the cfu/mL activated culture in an MRS liquid culture medium according to 2-4 vol%, culturing for 16-24h, centrifuging at 4 ℃ for 10-15min at 4000r/min, collecting precipitates, washing with sterile water for 3 times, and centrifuging at 4000r/min for 15-30min to obtain Lactobacillus fermentum ZS40 strain; the lactobacillus fermentum La obtained aboveAdding the Lactobacillus fermentumZS40 strain into 100mL of aqueous solution containing 0.08g/mL skimmed milk powder and 0.08g/mL glucose, mixing uniformly, and freeze-drying to obtain the Lactobacillus fermentum ZS40 leavening agent.
The invention has the beneficial effects that:
(1) the invention provides Lactobacillus fermentum ZS40, which can effectively improve ulcerative colitis and enrich Lactobacillus strain resources.
(2) The Lactobacillus fermentum ZS40 has the characteristics of bile salt resistance and gastric acid resistance, and provides a certain theoretical basis for the development of Lactobacillus fermentum ZS 40.
(3) The Lactobacillus fermentum ZS40 and the Staphylococcus xylosus ATCC29971 are mixed to be used as a leavening agent and applied to the preparation of the fermented sausage, so that the flavor and the quality of the fermented sausage are effectively improved. The Staphylococcus sacchari (Staphylococcus xylosus) ATCC29971 can release lipase and protease, enhance the flavor of meat products, generate nitrite reductase and improve the color of the products; the Lactobacillus fermentum ZS40 has the characteristics of high acid production speed, salt resistance, bacteriostasis and the like, can shorten the fermentation period and improve the food safety. The fermented sausage prepared by mixing Staphylococcus xylosus (Staphylococcus xylosus) ATCC29971 and Lactobacillus fermentum (Lactobacillus fermentum) ZS40 remarkably improves the number of lactic acid bacteria at the initial stage of fermentation and improves the fermentation capacity. The nitrite content and the TBA-N value are obviously reduced, and the sausage spoilage probability is reduced. The good flavor not only can endow the sausage with good quality, but also can increase the purchasing desire of consumers. Meanwhile, the edible safety of the meat products also attracts wide attention. The method is particularly important for improving the content of good flavor substances of the sausage and controlling the content of harmful biogenic amine in the sausage.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments will be briefly introduced below.
FIG. 1 shows the colony morphology of Lactobacillus fermentum ZS40 and the gram staining results of Lactobacillus fermentum ZS40 (A: the colony morphology of Lactobacillus fermentum ZS 40; B: the gram staining results of Lactobacillus fermentum ZS 40).
Figure 2 change in colon length (p <0.01 vs. DSS group, p < 0.0001).
FIG. 3 Colon pathology observations in different groups of mice.
Figure 4 levels of cytokines IL-1 β, IL-6, IL-10, TNF- α in the sera of different groups of mice (p <0.05, p <0.01, p <0.001, p <0.0001, compared to DSS group).
Figure 5 levels of serum superoxide dismutase (T-SOD), Myeloperoxidase (MPO), Catalase (CAT) and Malondialdehyde (MDA) in different groups of mice (p <0.05, p <0.01, p <0.001, p <0.0001, compared to DSS group).
FIG. 6 colon IkB-alpha, NF-kappa B, IL-6 and IkB-alpha mRNA and protein expression levels of different groups of mice; wherein, A: mRNA expression; b: protein expression (p <0.05, p <0.01, p <0.001, p <0.0001, in comparison to DSS group).
FIG. 7 different groups of mice colon p38 and JNK1/2mRNA and protein expression levels; wherein, A: mRNA expression; b: protein expression (p <0.05, p <0.01, p <0.001, p <0.0001, in comparison to DSS group).
Deposit description
The strain name is as follows: lactobacillus fermentum
Latin name: lactobacillus fermentum
The strain number is as follows: ZS40
The preservation organization: china general microbiological culture Collection center
The preservation organization is abbreviated as: CGMCC (China general microbiological culture Collection center)
Address: xilu No.1 Hospital No. 3 of Beijing market facing Yang district
The preservation date is as follows: 15/07/2019
Registration number of the preservation center: CGMCC No.18226
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings. All experiments were approved by the animal ethics committee of the university of Chongqing medical (Chongqing, China).
Lactobacillus bulgaricus (LDSB; AB200048) used in this experiment was purchased from China Center for Type Culture Collection (CCTCC) and used as a positive control.
0.35% pepsin, purchased from beijing solibao.
Biochemical kit, purchased from the institute of Engineer bioengineering, Chengcheng, Nanjing.
ELISA kit instruction, purchased from Nanjing to build Biotechnology Co.
Trizol reagent, purchased from semer fly.
Revert air First Strand cDNA Synthesis Kit, purchased from Saimei Fei.
A micro-spectrophotometer was purchased from Hangzhou Oerson instruments, Inc.
Real-time PCR instrument, purchased from semer fly.
PIPA lysate, purchased from semer fly.
BCA kit instructions, purchased from solibao.
Microplate reader, purchased from semer fly.
SDS-PAGE, purchased from Saimer fly.
Upright microscope, purchased from olympus scientific instruments ltd, guangdong.
Horseradish peroxidase conjugated secondary antibody, purchased from semer fly.
Chemiluminescent Liquid (ECL), purchased from semer fly.
Constant temperature shake incubators, purchased from schnakai instruments & equipment (shanghai) ltd.
Staphylococcus xylosus (Staphylococcus xylosus) ATCC29971, purchased from Hakka biosciences, Inc., Shanghai.
Sodium nitrite, food grade, CAS: 7632-00-0, purity: 99% from Hubei Xin run chemical Co., Ltd.
Sodium nitrate, food grade, CAS: 7631-99-4, purity: 99% from Asahi Biotech Limited in Anhui.
The ginger powder and the pepper powder are both food-grade products sold in the market.
Glucose, food grade, CAS: 50-99-7, purity: 99% from Hubei Xin run chemical Co., Ltd.
Monosodium glutamate, lotus monosodium glutamate produced by Henan lotus health industry, GmbH.
Salt, which is a natural salt produced by salt industry of Shanghai city and added with iodine.
Beef extract, the content of which is more than or equal to 99 percent, is purchased from JinsenSenyuan chemical Co., Ltd.
Casein peptone, CAS No.: 91079-40-2, food grade, purity: 99% from shanghai eosin industries ltd.
Yeast powder, CAS No.: 119-44-8, food grade, purity: 99% of the total amount of the food products purchased from Zhengzhou Yu and food additives Co.
Glucose, CAS No.: 50-99-7, food grade, purity: 99% from Hubei Xin run chemical Co., Ltd.
Sodium acetate, CAS No.: 6131-90-4, pH 0.5, food grade, sodium acetate content 99%, from Hubei Xin run German chemical Co.
Diammonium hydrogen citrate, CAS number: 3012-65-5, purity: 99% in food grade, purchased from Xinyang chemical Co., Ltd, Ningxiang.
Agar, CAS No.: 9002-18-0, food grade, agar substance content: 99% of the total amount of the products purchased from Zhengzhou Yi-source chemical products Co.
The MRS liquid medium described in the examples was: casein peptone 10.0g, beef extract 10.0g, yeast powder 5.0g, glucose 5.0g5.0g of sodium acetate, 2.0g of diammonium citrate, 801.0 g of Tween and K2HPO4 2.0g，MgSO4·7H2O 0.2g，MnSO4·H2O 0.05g，CaCO320.0g, agar 15.0g, water 1.0L, pH6.8, sterilization conditions: 121 ℃ for 15 min.
Example 1
Separation and identification of Lactobacillus fermentum ZS40
1. Isolation of Lactobacillus fermentum ZS40
Lactobacillus fermentum ZS40 was isolated from traditionally fermented yak yogurt in Basekuksk grassland, Shosu county.
Sucking 1mL of sample (traditional fermented yak milk in Basekuk grassland of Shosu county) by aseptic technique, and diluting with aseptic normal saline by 10 times gradient to 10 times-6. Selecting diluent with proper gradient, sucking 100 microliter of diluent by a sterile gun head, respectively spreading the diluent in MRS solid plate culture medium, culturing for 48h at 37 ℃, and observing and recording colony morphology. And selecting colonies with different forms on the plate for streaking separation, culturing at 37 ℃ for 48h, then selecting single colonies with different forms on the plate again for streaking separation, and repeating the steps for 2 to 3 times until pure single colonies with consistent forms are obtained.
The colony morphology of the strain with the number ZS40 is shown in figure 1, and the colony color is mostly white or milky white, the shape is round, the edge is neat, and the surface is wet and smooth.
2. Preliminary identification of Lactobacillus fermentum ZS40
The pure colonies on the plate were picked and inoculated in 5mL MRS liquid medium and cultured at 37 ℃ for 24 h. And (3) putting 1mL of the culture medium containing the bacteria into a sterile centrifuge tube, centrifuging for 10min at 4000r/min, removing an upper culture medium, suspending the thallus precipitate in sterile normal saline, performing gram stain microscopy, and preliminarily identifying the positive thallus precipitate as the lactobacillus.
The gram staining of the strain with the number ZS40 is bluish purple, namely the gram of the strain with the number ZS40 is positive, the cell morphology of the strain is shown in figure 1 under 100 times of oil lens, the cell morphology has long rods and short rods, and no budding is generated.
3. Molecular biological identification of strains
The genomic DNA of the above-mentioned Lactobacillus fermentum ZS40 was extracted using the kit, and the 16S rRNA sequence was amplified using PCR. Wherein the upstream primer 27F (5'-AGA GTT TGA TCC TGGCTC AG-3') 1. mu.L, the downstream primer 1495R (5'-CTA CGG CTA CCTTGT TAC GA-3') 1. mu.L, 2 XTaq plus Buffer 12.5. mu.L, and the template DNA 1. mu.L, were made up to 25. mu.L with sterile dd H2O. And sterile ultrapure water was used as a negative control instead of the template DNA. The amplification conditions were: 94 ℃ for 5 min; 94 ℃, 30s, 55 ℃, 30s, 72 ℃, 1min for 29 cycles, and finally an extension at 72 ℃ for 5 min.
Then 5 mul of amplification product is taken to carry out agarose gel electrophoresis detection, the agarose concentration is 1.5%, the electrophoresis condition is 110V, and 45 min. The PCR products which are successfully detected are sent to Beijing Optimalaceae biotechnology limited for sequencing, and the sequences which are successfully sequenced are compared and analyzed by using NCBI net (https:// blast.
By comparison, ZS40 strain Lactobacillus fermentum is identified, which is classified and named as Lactobacillus fermentum, and the strain is preserved in China general microbiological culture Collection center (CGMCC for short, address: No.1 Hospital No. 3, North Kyoho district) in 2019, 15.07.15 days, and the preservation number is CGMCC NO. 18226.
4. In vitro screening of Lactobacillus fermentum ZS40
(1) Lactobacillus fermentum ZS40 ability to tolerate 0.3% bile salts
Inoculating activated Lactobacillus fermentum ZS40 into MRS-THIO (MRS medium containing 0.2 wt% sodium thioglycolate) medium with bile salt content of 0.0 wt% and 0.3 wt% respectively at 2 vol%, culturing at 37 deg.C for 24 hr, loading blank medium and inoculated medium into 96-well plate (200 μ L per well) with blank MRS-THIO medium as control, and measuring OD of the above culture medium with different concentrations600nmThe value is obtained. The tolerance of the strain to bile salts is calculated according to the formula (1):
the results show that the strain with the number ZS40 can grow slowly in 0.3 wt% of bile salt, and the growth efficiency reaches 15.31% of that of the bile salt-free culture.
(2) Resistance test of Lactobacillus fermentum ZS40 to artificial gastric juice at pH3.0
Preparing artificial gastric juice: consists of 0.2 wt% NaCl and 0.35 wt% pepsin, the pH is adjusted to 3.0 by 1mol/LHCl, and then the mixture is filtered and sterilized by a filter membrane with the pore diameter of 0.22 mu m for standby.
Sucking 5mL of cultured bacteria-containing culture medium in a super-clean workbench, centrifuging for 10min at 3000r/min in a 10mL sterile centrifuge tube, removing an upper culture medium, collecting thalli, adding 5mL of sterile physiological saline, uniformly mixing to prepare a bacterial suspension, uniformly mixing 1mL of bacterial suspension with 9mL of artificial gastric juice with the pH of 3.0, treating 1mL of mixed solution as the artificial gastric juice for 0h of sample, and culturing the rest 9mL of mixed solution in a constant-temperature water bath shaker (37 ℃, 150r/min) for 3 h. And respectively sampling at 0h and 3h, respectively diluting the samples by 10-fold gradient, selecting proper gradient, determining viable count by adopting a plate coating method, culturing for 48h at 37 ℃ on an MRS solid culture medium, and calculating the survival rate (%):
the result shows that the strain with the number of ZS40 has better gastric acid resistance, and the survival rate of the strain after being treated by artificial gastric juice with the pH value of 3.0 for 3 hours reaches 79.32 percent.
The results show that the lactobacillus fermentum ZS40 has good in vitro resistance and can be used for subsequent animal experiments.
Secondly, improving effect of Lactobacillus fermentum ZS40 on colitis
1. Laboratory animal
SPF grade 7 week old male C57BL/6J mice, 50, were purchased from Chongqing university of medicine laboratory animal center. The animals were kept in a standardized laboratory at 25 + -2 deg.C, 50 + -5% relative humidity, 12h light and 12h dark, and the experiment was started after one week of acclimatization.
2. Establishment of animal model
7 days after acclimation, mice were randomized into 5 groups of 10 mice each: control group, DSS group, Sulfasalazine group (SSZ, 500mg/kg), Lactobacillus fermentum (Lactobacillus fermentum) ZS40 group (ZS40, 1.0 × 10)9CFU/mL) and Lactobacillus bulgaricus group (LB, 1.0X 10)9CFU/mL)。
The specific treatment method comprises the following steps:
(1) throughout the experiment, the Control group received standard food and drinking water and was gavaged daily with 0.1mL/10g of normal saline (mouse body weight);
(2) the DSS group received standard food and drinking water, drinking water was changed to 3% DSS solution in the third week, and the experiment period was gavaged daily with 0.1mL/10g normal saline (mouse body weight);
(3) the corresponding samples were gavaged daily after the start of the experiment in the SSZ, ZS40 and LB groups of mice and drinking water was replaced with 3 wt% DSS solution at the third week of the experiment. During the trial, gavage was performed daily and body weight was recorded and the entire experiment was completed at week 5.
3. Collection of samples
After 5 weeks, mice were sacrificed by cervical dislocation, blood from the eye was collected in a centrifuge tube at 4 ℃, 4000rpm/min, centrifuged for 10 minutes, and the supernatant was collected, sub-packaged in 200 μ L centrifuge tubes, and stored in a-80 ℃ refrigerator (Thermo Fisher Scientific co. ltd., Shanghai, China). After blood collection, the mouse was dissected, the colon was removed and the length and weight of the colon were measured and recorded by photographing, about 0.5cm of colon tissue was excised, fixed with 10 wt% formalin solution, and the remaining colon was frozen with liquid nitrogen and frozen in an ultra-low temperature freezer at-80 ℃ for use.
4. Colon length measurement in mice
DSS-induced colitis in mice results in colon shortening, so colon length is one of the important indicators for evaluating the severity of inflammation in colitis mice.
The colon length of each group of mice is shown in table 1. In the research, the 3 wt% DSS is used for constructing the mouse colitis, wherein the modeling principle of the 3 wt% DSS is that macromolecules enter intestinal mucosa tissues to cause tissue damage by destroying the intestinal mucosa and changing the permeability of the intestinal mucosa. And the configuration method is convenient and simple to operate, has good repeatability and is very similar to the diseased state of the human colitis. In the molding process, the colitis mouse has a weight reduction trend, after dissection, the colon is obviously shortened through comparison, and phenomena such as edema, hemorrhage, small ulcer and the like occur. To assess the effect of colitis on colon length in mice, we measured colon length results for all mice as shown in figure 2.
TABLE 1 Colon Length for groups of mice
| Grouping | Colon length/cm |
| Control group | 7.43±0.11 |
| DSS group | 4.83±0.38 |
| Group SSZ | 7.15±0.08 |
| ZS40 group | 6.90±0.22 |
| LB group | 5.55±0.07 |
Colon length was significantly longer in SSZ group, ZS40 group and LB group than in DSS group. Compared with the Control group, the DSS group mice have obviously shortened colon length and are accompanied with phenomena of congestion, edema and the like of intestinal mucosa. The colon of the mice treated by SSZ, ZS40 and LB can be treated to shorten the colon and obviously relieve the congestion, edema and ulcer. In this study, Lactobacillus fermentum ZS40 was able to alleviate colon shortening and inflammatory lesions in colon tissue with similar effect to sulfasalazine, a commonly used drug for colitis treatment.
5. Mouse colon tissue slice observation
Colon tissue was stained with hematoxylin-eosin (HE). The whole staining process comprises fixing, embedding, slicing, tabletting, dewaxing, staining and finally fixing the slices by neutral glue. Colonic morphological assessment was performed based on inflammation, crypt damage, mucosal damage. The colon histopathological morphology was observed and photographed using an upright microscope (Guangdong Olympus scientific instruments, Inc.).
The colon tissue section of the mouse stained with hematoxylin-eosin is taken, and the change of the tissue morphology is observed under an optical microscope. The results are shown in FIG. 3, Control group: the epithelial cells of the colon mucous membrane are complete, inflammatory cells are normal and have no infiltration, goblet cells are regularly arranged, and congestion and edema conditions are avoided; and (4) DSS group: epithelial cell damage is obvious, the intestinal wall is thickened, edema appears, inflammatory cell infiltration is realized, and goblet cells are reduced; after the treatment of SSZ, ZS40 and LB, although erosion still exists, congestion edema and cell infiltration are relieved, goblet cells are arranged regularly relative to DSS group, and the effect of improving colon tissues by Lactobacillus (Lactobacillus fermentum) ZS40 is most obvious, which shows that Lactobacillus fermentum ZS40 can improve DSS-induced colon injury and has promotion effect on preventing colitis.
6. Determination of IL-1 beta, IL-6, IL-10 and TNF-alpha levels in mouse serum
The collected plasma was centrifuged at 4000r/min at 4 ℃ for 10min, and the supernatant was collected. The levels of cytokines IL-1 β, IL-6, IL-10 and TNF- α in mouse serum were determined according to ELISA kit instructions (Nanjing Biotechnology Co., Ltd.).
Cytokines are small molecule proteins that transmit signals, which primarily modulate immune responses, mediate inflammatory responses, and participate in tissue repair, among others. There are two broad classes of cytokines involved in inflammatory responses, one class being anti-inflammatory cytokines, including IL-10 and the like; another class is pro-inflammatory cytokines including IL-1 β, IL-6, TNF- α, and the like. Studies have shown that during the development of inflammation, neutrophil granulocytes and macrophages infiltrate the colonic mucosa and secrete large amounts of pro-inflammatory cytokines and inhibit the secretion of anti-inflammatory cytokines, thus exacerbating the inflammatory state. IL-1 β is generally produced by mononuclear macrophages, but nucleated cells such as neutrophils and plasma cells can be synthesized and secreted after being stimulated by external antigens, and functions to promote activation and aggregation of inflammatory cells, increase permeability of epithelial cells and endothelial cells, aggravate intestinal mucositis, and mediate pain-sensitivity generation in inflammation. IL-6 is a proinflammatory factor produced by activated T cells and fibroblasts, can activate NF-kB signal channels to promote the expression of intercellular adhesion molecules, and the overexpression of the proinflammatory factor can influence the secretion of electrolytes of intestinal epithelial cells and change the permeability of the intestinal epithelial cells, so that neutrophils on mucous membranes are infiltrated to inflammation sites, and further the intestinal inflammatory response is induced or aggravated. TNF-alpha is produced by monocytes and macrophages, occurs earliest in the inflammatory response, and has the effect of increasing vascular endothelial cell permeability and aggregating inflammatory cells at the site of inflammation, causing inflammatory cell infiltration and causing tissue edema, which, when co-acting with the pro-inflammatory cytokine IFN-gamma, can result in structural changes in the intestinal epithelial cells. IL-10 is secreted by macrophages, dendritic cells and T cells, plays an important role in maintaining intestinal homeostasis, and has the main physiological functions of inhibiting neutrophils and reducing the expression of proinflammatory factors to protect tissues and organs from damage.
The levels of IL-1 β, IL-6, IL-10 and TNF- α in mouse serum were determined according to the instructions of the conventional biochemical kit and the results are shown in FIG. 4. The mice in the DSS group have obviously increased secretion of proinflammatory cytokines IL-1 beta, IL-6 and TNF-alpha, and the mice in the Control group have obviously reduced secretion of anti-inflammatory cytokines IL-10, but the contrary is true. After the intervention of ZS40 with Lactobacillus fermentum, the secretion of IL-1 beta, IL-6, IL-12 and TNF-alpha is inhibited, the secretion of IL-10 is promoted, and the level of mouse serum cytokines is closer to that of a normal group. The Lactobacillus fermentum ZS40 is proved to obviously inhibit the production of proinflammatory factors and promote the secretion of anti-inflammatory cytokines, thereby achieving the effect of reducing inflammation.
7. Determination of T-SOD, MPO, CAT and MDA in mouse serum
The collected plasma was centrifuged at 4000rpm/min at 4 ℃ for 10min, and the supernatant serum was collected. The activity of total superoxide dismutase (T-SOD; kit number A007-1-1), myeloperoxidase (MPO; A044) and catalase (CAT; A003-1-2) in mouse serum and the content of malondialdehyde (MDA; A001-1-2) were determined according to the instructions of a biochemical kit (Nanjing Jiancheng Chengcheng bioengineering institute, Nanjing).
The oxidative stress effect of ZS40 on colitis mouse serum was evaluated by monitoring antioxidant markers and biomarkers of lipid peroxidation, such as T-SOD, CAT, MPO and MDA. Total superoxide dismutase (T-SOD) exists in an antioxidant enzyme system, and can stabilize cell membranes by scavenging free radicals and inhibiting peroxidation, and the activity of the total superoxide dismutase (T-SOD) is an important index reflecting the anti-inflammatory reaction of organisms. Catalase (CAT) is an oxygen active scavenger, whose activity clearly reflects the ability of the body to scavenge hydroxyl radicals. Myeloperoxidase (MPO) is an enzyme present in neutrophils, and its activity is an important indicator of neutrophil infiltration and also of the severity of colitis. Malondialdehyde (MDA) is a lipid peroxide, formed as a result of free radical attack on the biofilm, which may reflect the degree of lipid peroxidation and thus colitis.
The contents of superoxide dismutase (T-SOD), Myeloperoxidase (MPO), Catalase (CAT) and Malondialdehyde (MDA) in colon tissues of mice were determined according to the instructions of a conventional biochemical kit, and the results are shown in FIG. 5. The T-SOD and CAT activities in the serum of the mice in the DSS group are the lowest, and the MPO and MDA contents are the highest; the Control group showed the opposite trend. In the study, SSZ, ZS40 and LB can up-regulate the activities of T-SOD and CAT, reduce the activity of MPO and the content of MDA, and show that the medicine can clear hydroxyl radicals, thereby having relieving effect on the colitis of mice. Among them, ZS40 group showed the best effect of the T-SOD, CAT, MPO and MDA in the serum, which are closest to the normal group. The results show that Lactobacillus fermentum ZS40 can improve the activity of antioxidant enzyme, improve the scavenging ability of organism to hydroxyl free radical, and finally reduce the damage of oxidative stress reaction to mouse colon.
8. Real-time fluorescent quantitative PCR (polymerase chain reaction) determination of mouse liver
Total RNA was extracted according to Trizol reagent (Samefei), reverse-transcribed into cDNA according to Revert air First Strand cDNA Synthesis Kit (Samefei), the concentration and purity of RNA and cDNA were measured with a micro spectrophotometer, and amplified with a real-time PCR instrument. The amplification conditions were: the reaction was carried out for 40 cycles at 95 ℃ for 15s, 60 ℃ for 30s, and 95 ℃ for 35 s. Finally, beta-actin is taken as an internal reference gene, and the expression level of each gene passes through 2-ΔΔCTAnd (4) calculating.
TABLE 2 primer sequences
8. Protein immunoassay in mouse colon
The colon samples stored at-80 ℃ were removed, total protein in the tissue was extracted using PIPA lysate (semefet) and the protein concentration was quantified in an enzyme reader (semefet) according to the BCA kit instructions (solibao). Protein samples were electrophoresed on SDS-PAGE (Saimer fly) and then transferred onto PVDF membrane. The transferred membrane was blocked with 5% skim milk for 1h at room temperature (25 ℃, 75 rpm; constant temperature shake incubator, Shidukai Instrument Co.Ltd., Shanghai, China) and then incubated with primary antibody overnight at 4 ℃ at the following dilution ratios of antibody: beta-actin (1: 500), nuclear factor kappa B (NF-kappa B p 65; 1: 1000), nuclear factor kappa B arrestin alpha (I kappa B-alpha; 1: 500), tumor necrosis factor alpha (TNF-alpha; 1: 500), p38 (1: 500), p-p38 (1: 1000), JNK1/2 (1: 500), and p-JNK1/2 (1: 500). After 3 washes with TBST buffer, the blot was incubated with horseradish peroxidase-conjugated secondary antibody (sermoex) for 1 hour at room temperature (37 ℃, 75rpm) and the immune complex was detected using chemiluminescence solution (ECL, sermoex), and finally observed and photographed with a multifunctional imager. The images were analyzed using Image J software (National Institutes of Health, Bethesda, Md.), and the relative expression level of the target protein was calculated using β -actin as an internal reference protein.
NF-. kappa.B and MAPK are two important pathways involved in the regulation of inflammatory processes. NF-. kappa.B plays an important role in the regulation of many immune and inflammatory responses. NF-. kappa.B normally binds to its suppressor IkB in the cytoplasm as an inactive dimer. Under the stimulation of inflammation, the activation of nuclear factor kappa B can generate inhibition protein kinase, so that I kappa B is phosphorylated and dissociated. NF-. kappa.B will be activated after separation from IkB.alpha.and transferred to the nucleus, which promotes induction and secretion of a large number of proinflammatory cytokines (e.g.: TNF-. alpha., IL-6), where TNF-. alpha.and IL-6 transcription has been shown to be affected by the NF-. kappa.B pathway, and furthermore, overexpression of TNF-. alpha.can activate NF-. kappa.B and exacerbate the inflammatory process. MAPK is an intracellular class of serine/threonine protein kinases that includes three subtypes: ERK, p38 and JNK. p38 mediates inflammation and apoptosis, and can transfer signal from cytoplasm to nucleus after activation, thereby promoting cell metabolism and cell survival. JNK is a class of mitogens, also known as stress-activated protein kinases or c-Jun amino-terminal kinases, which are activated by a variety of stimuli and play an important role in apoptosis, and JNK1/2 is an important member of the MAPK. In addition, JNK1/2 and p38 phosphorylation are major factors in regulating inflammatory cytokine expression. Growth factors, cytokines, inflammation and stress can activate MAPK signaling pathways and participate in the regulation and control processes of proliferation, differentiation, metastasis, apoptosis, cell cycle, inflammatory reaction and the like of cells. Research shows that NF-kappa B is one of the downstream components in MAPK signal transduction pathway, so that the activation of MAPK signal pathway can indirectly activate NF-kappa B signal pathway to induce the generation of inflammatory factors.
To investigate whether inhibition of colitis by Lactobacillus fermentum ZS40 was mediated by the NF-. kappa.B pathway, the inventors determined the relative expression levels of I.kappa.B-. alpha.NF-. kappa.Bp 65, IL-6 and TNF-. alpha.mRNA and protein in colon tissue, and the results are shown in FIG. 6. Compared with the Control group, the relative expression levels of NF-kappa Bp65, IL6 and TNF-alpha mRNA and protein in the colon tissue of the DSS group are increased, and the relative expression levels of I kappa B-alpha mRNA and protein are reduced, while the relative expression levels of Lactobacillus fermentum ZS40 are opposite and are closer to the Control group.
Further investigating whether inhibition of colitis by Lactobacillus fermentum ZS40 is mediated by MAPK pathway, the inventors determined the relative expression levels of p38 and JNK1/2mRNA and p38, p-p38, JNK1/2 and p-JNK1/2 protein in colon tissue, and the results are shown in FIG. 7. Compared with the Control group, the relative expression amounts of p38 and JNK1/2mRNA and p38, p-p38, JNK1/2 and p-JNK1/2 protein in the colon tissue of the DSS group are increased, and the expression is obviously inhibited by Lactobacillus fermentum ZS 40. In conclusion, the down-regulation of the expression of inflammation-related genes and proteins by blocking the activation of NF- κ B and MAPK signaling pathways is an ideal approach in the treatment of colitis.
Example 2
A fermented sausage comprises the following preparation steps:
(1) mincing 100 parts by weight of fresh pork without fascia and tendon, and stirring to prepare pork stuffing;
(2) adding auxiliary materials into the pork stuffing obtained in the step (1) and pickling at 4 ℃;
(3) inoculating 0.2 part by weight of leavening agent into the pork stuffing obtained in the step (2), and uniformly stirring to prepare the sausage;
(4) fermenting the sausage obtained in the step (3) at 25 ℃ and RH 95% for 3 days;
(5) fermenting the sausage in the step (4) at 15 ℃ and RH 72% for 11 days;
(6) vacuum packaging to obtain fermented sausage;
the leavening agent is a Lactobacillus fermentum ZS40 leavening agent;
the preparation method of the Lactobacillus fermentum ZS40 leavening agent comprises the steps of inoculating Lactobacillus fermentum ZS40 into an MRS liquid culture medium, culturing for 16h at 37 ℃ and activating to obtain the Lactobacillus fermentum ZS40 containing Lactobacillus fermentum with the concentration of 1 multiplied by 106cfu/mL activated culture; the Lactobacillus fermentumZS40 containing Lactobacillus fermentum is 1 × 106Inoculating the cfu/mL activated culture into an MRS liquid culture medium according to 2 vol%, culturing for 16h, centrifuging at 4 ℃ and 4000r/min for 15min, collecting precipitates, washing with sterile water for 3 times, and centrifuging at 4000r/min for 20min to obtain Lactobacillus fermentum ZS40 strain; adding the Lactobacillus fermentum ZS40 strain into 100mL of aqueous solution containing 0.08g/mL of skim milk powder and 0.08g/mL of glucose, mixing uniformly, and freeze-drying to obtain the Lactobacillus fermentum ZS40 leavening agent.
The auxiliary materials comprise 2 parts by weight of salt, 0.2 part by weight of monosodium glutamate, 1 part by weight of glucose, 0.1 part by weight of pepper powder, 0.1 part by weight of ginger powder, 0.001 part by weight of sodium nitrite and 0.01 part by weight of sodium nitrate;
example 3
Essentially the same as example 2, except that:
the leaven is Staphylococcus xylosus (Staphylococcus xylosus) ATCC29971 leaven.
The preparation method of the Staphylococcus xylosus (Staphylococcus xylosus) ATCC29971 leavening agent comprises the steps of inoculating Staphylococcus xylosus (Staphylococcus xylosus) ATCC29971 into an MRS liquid culture medium, culturing at 37 ℃ for 16 hours, and activating to obtain Staphylococcus xylosus (Staphylococcus xylosus) ATCC29971 with the volume of 1 x 106cfu/mL activated culture; the content of Staphylococcus xylosus (Staphylococcus xylosus) ATCC29971 was 1X 106Inoculating the cfu/mL activated culture into an MRS liquid culture medium according to 2 vol%, culturing for 16h, centrifuging at 4 ℃ at 4000r/min for 15min, collecting precipitates, washing with sterile water for 3 times, and centrifuging at 4000r/min for 20min to obtain Staphylococcus xylosus (Staphylococcus xylosus) ATCC29971 strain; mixing the obtained xylose grapeAdding 100mL of aqueous solution containing 0.08g/mL of skim milk powder and 0.08g/mL of glucose into a coccus (Staphylococcus xylosus) ATCC29971 strain, uniformly mixing, and freeze-drying to obtain the Staphylococcus xylosus ATCC29971 starter.
Example 4
Essentially the same as example 2, except that:
the leavening agent is a Staphylococcus xylosus (Staphylococcus xylosus) ATCC29971 leavening agent and a Lactobacillus fermentum (Lactobacillus fermentum) ZS40 leavening agent which are mixed according to the mass ratio of 2: 1;
the preparation method of the Staphylococcus xylosus (Staphylococcus xylosus) ATCC29971 leavening agent comprises the steps of inoculating Staphylococcus xylosus (Staphylococcus xylosus) ATCC29971 into an MRS liquid culture medium, culturing at 37 ℃ for 16 hours, and activating to obtain Staphylococcus xylosus (Staphylococcus xylosus) ATCC29971 with the volume of 1 x 106cfu/mL activated culture; the content of Staphylococcus xylosus (Staphylococcus xylosus) ATCC29971 was 1X 106Inoculating the cfu/mL activated culture into an MRS liquid culture medium according to 2 vol%, culturing for 16h, centrifuging at 4 ℃ at 4000r/min for 15min, collecting precipitates, washing with sterile water for 3 times, and centrifuging at 4000r/min for 20min to obtain Staphylococcus xylosus (Staphylococcus xylosus) ATCC29971 strain; adding the obtained Staphylococcus xylosus (Staphylococcus xylosus) ATCC29971 strain into 100mL of aqueous solution containing 0.08g/mL of skim milk powder and 0.08g/mL of glucose, uniformly mixing, and freeze-drying to obtain the Staphylococcus xylosus (Staphylococcus xylosus) ATCC29971 leavening agent.
The preparation method of the Lactobacillus fermentum ZS40 leavening agent comprises the steps of inoculating Lactobacillus fermentum ZS40 into an MRS liquid culture medium, culturing for 16h at 37 ℃ and activating to obtain the Lactobacillus fermentum ZS40 containing Lactobacillus fermentum with the concentration of 1 multiplied by 106cfu/mL activated culture; the Lactobacillus fermentumZS40 containing Lactobacillus fermentum is 1 × 106Inoculating the activated cfu/mL culture in MRS liquid culture medium at 2 vol%, culturing for 16h, centrifuging at 4 deg.C and 4000r/min for 15min, collecting precipitate, washing with sterile water for 3 times, and centrifuging at 4000r/min for 20min to obtain Lactobacillus fermentumfermentumZS40 strain; adding the Lactobacillus fermentum ZS40 strain into 100mL of aqueous solution containing 0.08g/mL of skim milk powder and 0.08g/mL of glucose, mixing uniformly, and freeze-drying to obtain the Lactobacillus fermentum ZS40 leavening agent.
Example 5
The method is basically the same as example 4, except that the leavening agent is Staphylococcus xylosus (Staphylococcus xylosus) ATCC29971 leavening agent and Lactobacillus fermentum (Lactobacillus fermentum) ZS40 leavening agent which are mixed according to the mass ratio of 3: 1.
Example 6
The method is basically the same as example 4, except that the leavening agent is Staphylococcus xylosus (Staphylococcus xylosus) ATCC29971 leavening agent and Lactobacillus fermentum (Lactobacillus fermentum) ZS40 leavening agent which are mixed according to the mass ratio of 1: 1.
Example 7
The method is basically the same as example 4, except that the leavening agent is Staphylococcus xylosus (Staphylococcus xylosus) ATCC29971 leavening agent and Lactobacillus fermentum (Lactobacillus fermentum) ZS40 leavening agent which are mixed according to the mass ratio of 1: 3.
Test example 1
The fermented sausages prepared in examples 2 to 7 were subjected to sensory evaluation. 20 background persons with food professional knowledge form a sensory evaluation group, and the color, smell, tissue state and taste of the fermented sausage are evaluated by the group members according to GB/T22210-2008. The scoring criteria are shown in table 1.
TABLE 1 sensory evaluation criteria for fermented sausages
TABLE 2 sensory evaluation of fermented sausages
| Colour(s) | Smell(s) | Tissue state | Taste of the product | Total score | |
| Example 2 | 16 | 16 | 20 | 22 | 78 |
| Example 3 | 14 | 15 | 20 | 20 | 39 |
| Example 4 | 20 | 20 | 26 | 28 | 94 |
| Example 5 | 18 | 18 | 22 | 23 | 81 |
| Example 6 | 18 | 20 | 25 | 27 | 90 |
| Example 7 | 18 | 20 | 24 | 25 | 87 |
As can be seen from Table 2, Staphylococcus xylosus (Staphylococcus xylosus) ATCC29971 can release lipase and protease, enhance flavor of meat products, produce nitrite reductase, and improve color of products; the Lactobacillus fermentum ZS40 has the characteristics of high acid production speed, salt resistance, bacteriostasis and the like, can shorten the fermentation period and improve the food safety. The fermented sausage prepared by mixing Staphylococcus xylosus (Staphylococcus xylosus) ATCC29971 and Lactobacillus fermentum (Lactobacillus fermentum) ZS40 remarkably improves the number of lactic acid bacteria at the initial stage of fermentation and improves the fermentation capacity. The nitrite content and the TBA-N value are obviously reduced, and the sausage spoilage probability is reduced. The good flavor not only can endow the sausage with good quality, but also can increase the purchasing desire of consumers. Meanwhile, the edible safety of the meat products also attracts wide attention. The method is particularly important for improving the content of good flavor substances of the sausage and controlling the content of harmful biogenic amine in the sausage.
In the whole fermentation process of the fermented sausage, products of growth and metabolism of microorganisms and various physical and chemical reaction products generate the special fragrance of the fermented meat product, and the taste and smell of the fermented sausage can be increased. However, Lactobacillus fermentum ZS40 and Staphylococcus xylosus ATCC29971 have different characteristics, and Lactobacillus fermentum ZS40 and Staphylococcus xylosus ATCC29971 with different proportions have different influences on the flavor and meat quality of the fermented sausage. Staphylococcus xylosus (Staphylococcus xylosus) ATCC29971 can decompose protein, fat and peptide, and Lactobacillus fermentum (Lactobacillus fermentum) ZS40 can not only utilize carbohydrate to produce lactic acid, but also can contribute to improving flavor by decomposing the activity of peptide.
Test example 2
The nitrite content was tested for examples 2-4. The nitrite content is tested according to GB/T5009.33-2010 determination of nitrite and nitrate in food. The test results are shown in Table 3.
TABLE 3 determination of nitrite content in fermented sausages
| Nitrite content/(mg/kg) | |
| Example 2 | 14.9 |
| Example 3 | 17.5 |
| Example 4 | 12.4 |
| Example 5 | 14.2 |
| Example 6 | 13.1 |
| Example 7 | 13.7 |
The nitrite influenced by nitrite in the fermented sausage has the functions of color development, oxidation resistance and corrosion prevention, the unpleasant flavor of raw meat can be eliminated, and the product quality is improved. However, the excessive residual quantity of the nitrosamine can generate nitrosamine with strong carcinogenic effect with biogenic amine in the sausage, which is a food safety problem which is concerned by the food industry at present. As can be seen from Table 3, in example 4 fermented sausages were prepared by adding Staphylococcus xylosus (Staphylococcus xylosus) ATCC29971 and Lactobacillus fermentum (Lactobacillus fermentum) ZS40 as leavening agents, the nitrite content was significantly lower than in examples 2 to 3, and the nitrite content in examples 2 to 7 was lower than the maximum allowable residual amount of 30mg/kg in national food safety regulations.
Test example 3
Examples 2-7 were tested for volatile base based total nitrogen (TVB-N). The testing of total volatile basic nitrogen (TVB-N) was performed with reference to G B/T5009.44-2003 "analytical methods for meat and meat product hygiene standards". The test results are shown in Table 4.
TABLE 4 testing of volatile base-based Total Nitrogen (TVB-N) in fermented sausages
| TVB-N value/(mg/kg) | |
| Example 2 | 8.3 |
| Example 3 | 11.6 |
| Example 4 | 5.9 |
| Example 5 | 7.8 |
| Example 6 | 7.1 |
| Example 7 | 7.5 |
TVB-N is volatile basic total nitrogen and is used for measuring the freshness of meat, and the smaller the TVB-N value is, the fresher the meat quality is. The fermented sausage prepared by mixing Staphylococcus xylosus (Staphylococcus xylosus) ATCC29971 and Lactobacillus fermentum (Lactobacillus fermentum) ZS40 remarkably improves the number of lactic acid bacteria at the initial stage of fermentation and improves the fermentation capacity. The TBA-N value is obviously reduced, and the sausage spoilage probability is reduced.
The foregoing detailed description of the preferred embodiments of the invention has been presented. It should be understood that numerous modifications and variations could be devised by those skilled in the art in light of the present teachings without departing from the inventive concepts. Therefore, the technical solutions available to those skilled in the art through logic analysis, reasoning and limited experiments based on the prior art according to the concept of the present invention should be within the scope of protection defined by the claims.
SEQUENCE LISTING
<110> university of Chongqing second teacher
<120> Lactobacillus fermentum ZS40 having preventive effect on colitis
<160> 3
<170> PatentIn version 3.5
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<211> 1474
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<213> Artificial Synthesis
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<223>Lactobacillus fermentum ZS40
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gatggctttg ctataatgca gtcgaacgcg ttggcccaat tgattgatgg tgcttgcacc 60
tgattgattt tggtcgccaa cgagtggcgg acgggtgagt aacacgtagg taacctgccc 120
agaagcgggg gacaacattt ggaaacagat gctaataccg cataacagcg ttgttcgcat 180
gaacaacgct taaaagatgg cttctcgcta tcacttctgg atggacctgc ggtgcattag 240
cttgttggtg gggtaacggc ctaccaaggc gatgatgcat agccgagttg agagactgat 300
cggccacaat gggactgaga cacggcccat actcctacgg gaggcagcag tagggaatct 360
tccacaatgg gcgcaagcct gatggagcaa caccgcgtga gtgaagaagg gtttcggctc 420
gtaaagctct gttgttaaag aagaacacgt atgagagtaa ctgttcatac gttgacggta 480
tttaaccaga aagtcacggc taactacgtg ccagcagccg cggtaatacg taggtggcaa 540
gcgttatccg gatttattgg gcgtaaagag agtgcaggcg gttttctaag tctgatgtga 600
aagccttcgg cttaaccgga gaagtgcatc ggaaactgga taacttgagt gcagaagagg 660
gtagtggaac tccatgtgta gcggtggaat gcgtagatat atggaagaac accagtggcg 720
aaggcggcta cctggtctgc aactgacgct gagactcgaa agcatgggta gcgaacagga 780
ttagataccc tggtagtcca tgccgtaaac gatgagtgct aggtgttgga gggtttccgc 840
ccttcagtgc cggagctaac gcattaagca ctccgcctgg ggagtacgac cgcaaggttg 900
aaactcaaag gaattgacgg gggcccgcac aagcggtgga gcatgtggtt taattcgaag 960
ctacgcgaag aaccttacca ggtcttgaca tcttgcgcca accctagaga tagggcgttt 1020
ccttcgggaa cgcaatgaca ggtggtgcat ggtcgtcgtc agctcgtgtc gtgagatgtt 1080
gggttaagtc ccgcaacgag cgcaaccctt gttactagtt gccagcatta agttgggcac 1140
tctagtgaga ctgccggtga caaaccggag gaaggtgggg acgacgtcag atcatcatgc 1200
cccttatgac ctgggctaca cacgtgctac aatggacggt acaacgagtc gcgaactcgc 1260
gagggcaagc aaatctctta aaaccgttct cagttcggac tgcaggctgc aactcgcctg 1320
cacgaagtcg gaatcgctag taatcgcgga tcagcatgcc gcggtgaata cgttcccggg 1380
ccttgtacac accgcccgtc acaccatgag agtttgtaac acccaaagtc ggtggggtaa 1440
ccttttagga gccagccgcc taaggtcaca gaag 1474
<210> 2
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Claims (7)
1. Lactobacillus fermentum ZS40 with effect in preventing colitis, wherein the Lactobacillus fermentum has been deposited at the China general microbiological culture Collection center (CGMCC) 7, 15 days 2019 with the deposit number of CGMCC No. 18226.
2. The lactobacillus fermentum ZS40 as claimed in claim 1, wherein the morphological characteristics are in particular:
the characteristics of the thallus are as follows: the gram stain is bluish purple, the bacterial strain is gram positive, the cell morphology has long rods and short rods, and no budding reproduction exists;
colony characteristics: the colony color is white or milk white, the shape is round, the edge is neat, and the surface is moist and smooth.
3. Lactobacillus fermentum ZS40, according to claim 1, wherein the lactobacillus fermentum ZS40 is characterized by: the lactobacillus fermentum ZS40 has gastric acid resistance.
4. The lactobacillus fermentum ZS40 of claim 3, wherein the lactobacillus fermentum ZS40 is characterized by: the survival rate of the artificial gastric juice treated by pH3.0 for 3 hours reaches 75-85 percent; the growth efficiency of the bacteria in 0.30 wt% of bile salt reaches 10-20 wt% of that of the bacteria cultured without bile salt.
5. The lactobacillus fermentum ZS40 of claim 4, wherein the lactobacillus fermentum ZS40 is characterized by: the survival rate of the artificial gastric juice treated by pH3.0 for 3 hours reaches 79.32 percent; the growth efficiency of the bacterium in 0.30% of bile salt reaches 15.31% of that in the bile salt-free culture.
6. Use of the lactobacillus fermentum ZS40 as defined in any one of claims 1 to 5 in the preparation of a food or/and a pharmaceutical product for preventing colitis.
7. The use of the lactobacillus fermentum ZS40 as defined in any one of claims 1 to 5 in fermenting meat products.
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| CN116270763A (en) * | 2023-04-04 | 2023-06-23 | 广东南芯医疗科技有限公司 | Application of lactobacillus fermentum E15 in preparation of medicines for preventing or treating intestinal inflammation |
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| CN114854647A (en) * | 2022-06-16 | 2022-08-05 | 广西大学 | Lactobacillus fermentum and culture and application thereof |
| CN114854647B (en) * | 2022-06-16 | 2023-08-29 | 广西大学 | Lactobacillus fermentum and culture and application thereof |
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| CN116836879A (en) * | 2023-07-21 | 2023-10-03 | 四川大学 | Lactobacillus fermentum with colon cancer cell growth inhibition effect and application thereof |
| CN116836879B (en) * | 2023-07-21 | 2024-01-23 | 四川大学 | Lactobacillus fermentum with colon cancer cell growth inhibition effect and application thereof |
| CN117535175A (en) * | 2023-10-12 | 2024-02-09 | 善恩康生物科技（苏州）有限公司 | Composite probiotics and application thereof in preparation of products for preventing or assisting in treating colorectal cancer |
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