WO2023159960A1 - 一株益生植物乳杆菌及其在低盐发酵肉食品制备中的应用 - Google Patents
一株益生植物乳杆菌及其在低盐发酵肉食品制备中的应用 Download PDFInfo
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- WO2023159960A1 WO2023159960A1 PCT/CN2022/122988 CN2022122988W WO2023159960A1 WO 2023159960 A1 WO2023159960 A1 WO 2023159960A1 CN 2022122988 W CN2022122988 W CN 2022122988W WO 2023159960 A1 WO2023159960 A1 WO 2023159960A1
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- lactobacillus plantarum
- meat
- cmrc
- low
- fermented
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Images
Classifications
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
- A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
- A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
-
- 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
- A23L13/00—Meat products; Meat meal; Preparation or treatment thereof
- A23L13/40—Meat products; Meat meal; Preparation or treatment thereof containing additives
- A23L13/45—Addition of, or treatment with, microorganisms
-
- 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
- A23L13/00—Meat products; Meat meal; Preparation or treatment thereof
- A23L13/60—Comminuted or emulsified meat products, e.g. sausages; Reformed meat from comminuted meat product
- A23L13/65—Sausages
-
- 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
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
-
- 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
- A23V2400/11—Lactobacillus
- A23V2400/169—Plantarum
Definitions
- the invention relates to the technical field of microorganisms, in particular to a probiotic plant lactobacillus and its application in the preparation of low-salt fermented meat food.
- Fermented meat food has the characteristics of long shelf life and unique flavor, and is deeply loved by consumers.
- the amount of sodium chloride added in the production of fermented meat is usually as high as 4-10%.
- Higher sodium chloride content is likely to cause high blood pressure and cardiovascular diseases in consumers, which is not conducive to human health.
- low-salt fermented meat products that can improve intestinal flora and promote human health are favored by more and more consumers.
- Lactic acid bacteria have an important impact on the safety, flavor and nutritional quality of low-salt fermented meat products.
- lactic acid bacteria can reduce the pH value and produce bacteriocins to inhibit the growth of harmful bacteria in low-salt fermented meat and ensure the microbial safety of low-salt fermented meat foods;
- lactic acid bacteria can metabolize carbohydrates, muscle protein and fat, and produce acids , esters, peptides and other flavor substances, thereby improving the sensory flavor quality of low-salt fermented meat products.
- probiotic lactic acid bacteria that can be used in low-salt fermented meat foods has also attracted more and more attention.
- probiotic lactic acid bacteria derived from the human intestine because these lactic acid bacteria are more likely to have probiotic properties such as gastric acid resistance and high intestinal adhesion ability.
- the meat fermentation performance of lactic acid bacteria derived from human intestines is often not ideal, and it is not suitable as a meat starter.
- lactic acid bacteria starters with excellent meat fermentation performance and probiotic performance are rarely reported.
- a single strain with excellent meat fermentation performance and probiotic performance as a starter is more conducive to reducing production costs.
- Chinese patent CN113957021A discloses a Lactobacillus plantarum grx401 with resistance to digestive stress and high intestinal adhesion ability and its application. The strain was reported to have excellent meat fermentation properties.
- Chinese patent CN108410774B discloses a compound bacterial agent and its application in functional fermented meat food, which uses a compound bacterial agent comprising Lactobacillus plantarum and Staphylococcus xylosus.
- Chinese patent CN113373097A discloses a strain of Lactobacillus plantarum and its application in the fermentation of existing Sichuan-flavored sausages, but it has not been reported that the strain has excellent probiotic properties, and what is used is Lactobacillus plantarum, Pediococcus pentosaceae and staphylococcus Compound bacteria.
- the object of the present invention is to provide a probiotic plant lactobacillus and its application in the preparation of low-salt fermented meat food.
- the present invention provides the following technical solutions:
- the present invention provides Lactobacillus plantarum (Lactobacillus plantarum) CMRC 19L, which was preserved on September 9, 2015 in the General Microbiology Center of China Microbiological Culture Collection Management Committee (CGMCC for short, address: No. 1, Beichen West Road, Chaoyang District, Beijing) No. 3, Institute of Microbiology, Chinese Academy of Sciences, Zip code 100101), the classification name is Lactobacillus plantarum, and the preservation number is CGMCC No.11347.
- CGMCC General Microbiology Center of China Microbiological Culture Collection Management Committee
- the 16S rDNA gene sequence of Lactobacillus plantarum CMRC 19L is shown in SEQ ID No.1.
- the invention provides a fermented product, which is prepared by fermentation and cultivation of Lactobacillus plantarum CMRC 19L.
- the fermented product comprises Lactobacillus plantarum CMRC 19L or comprises Lactobacillus plantarum CMRC 19L and fermentation supernatant thereof.
- the invention provides a bacterial agent, which contains Lactobacillus plantarum CMRC 19L.
- the present invention also provides a starter, which contains plantaractobacillus CMRC 19L.
- Lactobacillus plantarum CMRC 19L exists in the form of live bacteria in the above-mentioned bacterium agent and starter.
- the above bacterial agents and fermentation agents also contain a freeze-drying protectant.
- the above bacterial agent or starter can be liquid bacterial agent, starter or solid bacterial agent, starter (for example: dry powder bacterial agent).
- the invention provides a preparation method of the bacterial agent or the fermentation agent, and the method comprises the step of culturing Lactobacillus plantarum CMRC19L to obtain bacterial liquid.
- the culturing is carried out at 37°C.
- the medium used for cultivation includes but not limited to MRS broth medium and other commonly used medium for Lactobacillus plantarum.
- the bacterial liquid obtained above can be directly or added with the auxiliary materials allowed in the field of microbial preparations to prepare liquid bacterial agents or starters, and the bacterial cells in the bacterial liquid can also be collected and mixed with auxiliary materials allowed in the field of microbial preparations such as freeze-drying protective agents, and then vacuum-frozen Dried and prepared as dry powder fungus or starter.
- the live bacterial content of Lactobacillus plantarum CMRC 19L is 10 10 -10 12 CFU/g.
- Lactobacillus plantarum CMRC 19L has a fast growth rate and has obvious inhibitory effect on pathogenic bacteria such as E. coli and Staphylococcus aureus. It not only has excellent meat fermentation performance, but also can better ensure fermentation
- the food safety and flavor quality of meat food and can tolerate the gastrointestinal tract environment, and has high adhesion to intestinal mucus protein and intestinal epithelial cells, and has excellent probiotic properties.
- the present invention provides the application of Lactobacillus plantarum CMRC 19L or the bacterial agent or the starter in the preparation of fermented food;
- the fermented food is preferably fermented meat food; more preferably low-salt fermented meat food; most preferably low-salt fermented sausage.
- the pathogenic microorganism is preferably pathogenic Escherichia coli or Staphylococcus aureus.
- the product includes but not limited to food, feed, probiotic preparation or medicine.
- the invention provides a low-salt fermented meat food, which is prepared by fermenting raw materials containing livestock and poultry meat with Lactobacillus plantarum CMRC 19L or the bacteria agent or the starter agent.
- the inoculation amount of the fermentation is 10 9 -10 11 CFU Lactobacillus plantarum CMRC 19L/kg raw material. More preferably 10 10 CFU Lactobacillus plantarum CMRC 19L/kg raw material.
- the fermentation is carried out at 28-32°C and 90 ⁇ 3% humidity for 1-3 days, and then at 13-17°C and 80 ⁇ 3% humidity for 16-20 days.
- the fermentation is carried out at 30°C and 90 ⁇ 3% humidity for 2 days, and then at 15°C and 80 ⁇ 3% humidity for 18 days.
- the raw material comprises livestock and poultry meat and sodium chloride in an amount of 1-3% by weight of the livestock and poultry meat.
- the sodium chloride accounts for 1-2% by mass of the livestock and poultry meat.
- the livestock and poultry meat includes but not limited to pork, mutton, chicken or beef, and may include lean meat and/or fat.
- the raw materials also include spices and/or additives.
- the raw materials include the following components: lean meat 750-850g, fat 150-250g, sodium chloride 10-20g, black pepper 1-3g, white pepper 1-3g, glucose 5-15g, Sodium nitrite 0.05-0.1g, iso-VC sodium 0.1-1g, tripolyphosphate 1-3g, dry red wine 5-15g, fresh garlic 3-6g, water 15-25g.
- the present invention also provides a method for preparing low-salt fermented meat food.
- the method includes the step of fermenting raw materials containing livestock and poultry meat with Lactobacillus plantarum CMRC 19L or the bacterium or the starter.
- the inoculation amount of the fermentation is 10 9 -10 11 CFU Lactobacillus plantarum CMRC 19L/kg raw material. More preferably 10 10 CFU Lactobacillus plantarum CMRC 19L/kg raw material.
- the fermentation is carried out at 28-32°C and 90 ⁇ 3% humidity for 1-3 days, and then at 13-17°C and 80 ⁇ 3% humidity for 16-20 days.
- the fermentation is carried out at 30°C and 90 ⁇ 3% humidity for 2 days, and then at 15°C and 80 ⁇ 3% humidity for 18 days.
- the raw material comprises livestock and poultry meat and sodium chloride in an amount of 1-3% by weight of the livestock and poultry meat.
- the sodium chloride accounts for 1-2% by mass of the livestock and poultry meat.
- the livestock and poultry meat includes but not limited to pork, mutton, chicken or beef, and may include lean meat and/or fat.
- the raw material also contains spices and/or additives.
- the raw materials include the following components: lean meat 750-850g, fat 150-250g, sodium chloride 10-20g, black pepper 1-3g, white pepper 1-3g, glucose 5-15g, Sodium nitrite 0.05-0.1g, iso-VC sodium 0.1-1g, tripolyphosphate 1-3g, dry red wine 5-15g, fresh garlic 3-6g, water 15-25g.
- the method includes the steps of:
- the present invention provides a strain of Lactobacillus plantarum CMRC 19L, which is isolated from traditional naturally fermented sour meat, has a fast growth rate, can inhibit the growth of pathogenic bacteria, has excellent meat fermentation performance, and is resistant to In the gastrointestinal tract environment, it shows high adhesion to intestinal mucus protein and intestinal epithelial cells, and has good probiotic properties.
- the strain can ensure the edible safety and flavor quality of low-salt fermented meat, shorten the fermentation period, and significantly improve the nutritional value of fermented meat.
- Fig. 1 is the inhibitory effect of Lactobacillus plantarum CGMCC No.11347 culture supernatant to Escherichia coli O157:H7 and Staphylococcus aureus in Example 2 of the present invention, wherein, contrast bacterial strain CGMCC No.9741, CGMCC No.9742, CGMCCNo .11348 are Lactobacillus plantarum, all isolated from Guizhou traditional naturally fermented sour meat, the left picture shows the inhibitory effect of Staphylococcus aureus, and the right picture shows the inhibitory effect of Escherichia coli O157:H7;
- Fig. 2 is the adherent cell diagram (400 ⁇ ) of Lactobacillus plantarum CGMCC No.11347 in Example 2 of the present invention
- Fig. 3 is the detection result of the number of live bacteria of lactic acid bacteria in rat intestinal tract in the experimental example of the present invention, after respectively feeding basal feed (blank control), fresh pork, fermented sausage for 30 days and 60 days continuously, the live bacteria of lactic acid bacteria in intestinal tract of male and female rats number;
- Fig. 4 is the detection result of rat body weight gain in the experimental example of the present invention, after respectively feeding basal feed (blank control), fresh pork, fermented sausage continuously 30 days and 60 days, the body weight that male and female rats increase;
- male-30 days means that male rats were fed continuously for 30 days
- female-30 days means that female rats were fed continuously for 30 days
- male-60 days means that male rats were fed continuously for 30 days. Feeding for 60 days
- female-60 days means that the female rats were fed continuously for 60 days.
- Lactobacillus plantarum (Lactobacillus plantarum) CMRC 19L was preserved on September 9, 2015 in the General Microbiology Center of China Committee for Culture Collection of Microorganisms (CGMCC for short), and the preservation address is: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Zip code 100101), the deposit number is CGMCC No.11347.
- the control strain CGMCC No.9741 involved in the following examples has been preserved on September 28, 2014 in the General Microbiology Center of the China Committee for the Collection of Microbial Cultures (abbreviated as CGMCC, and the address is No. 1, Beichen West Road, Chaoyang District, Beijing. No., Institute of Microbiology, Chinese Academy of Sciences, Zip Code: 100101)
- CGMCC General Microbiology Center of the China Committee for the Collection of Microbial Cultures
- the preservation number is CGMCC No.9741, and the strain has been disclosed in the patent application CN 104560808 A.
- CGMCC No.9742 was deposited in the General Microbiology Center of China Committee for the Collection of Microbial Cultures on September 28, 2014. Postal code: 100101, the preservation number is CGMCCNo.9742, and the strain has been disclosed in the patent application CN 104560802A.
- CGMCC No.11348 was deposited on September 09, 2015 in the General Microbiology Center of China Committee for the Collection of Microbial Cultures. Zip code: 100101, deposit number is CGMCC No.11348.
- the strain has been disclosed in the patent application CN108410774A.
- Volatile flavor substances were detected with reference to the method provided in K.A.Gutsche, T.B.Tran, R.F.Vogel, Production of volatile compounds by Lactobacillus sakei from branched chainalpha-keto acids[J]. Food Microbiology, 2012, 29: 224-228. ( SPME-GC-MS method).
- the content of free amino acids is determined according to Li Pinglan, Shen Qingwu, et al. Effects of microbial enzymes and meat tissue enzymes on free amino acids in dry fermented sausages [J]. Food and Fermentation Industry, 2005, 31(5): 134-138. Methods.
- the Lactobacillus plantarum CMRC 19L provided by the present invention is isolated from Guizhou traditional naturally fermented sour meat: take 25g of sour meat sample, add it to 225mL sterilized physiological saline, beat for 8 minutes to homogenize. Take the supernatant, spread it on the MRS agar medium after serial dilution, and after culturing at 30°C for 48 hours, randomly pick a single colony with neat edges and smooth edges from the plate with the highest dilution for further culture and purification.
- strains were subjected to physical and chemical determinations such as morphology, salt resistance, nitrite resistance, glucose homotype/heterotype fermentation, and Gram staining was selected as positive and resistant to 8% sodium chloride and 0.015% sodium nitrite , homozygous or facultative heterofermentative strain CMRC 19L for further identification.
- strain CMRC 19L The survival rate of strain CMRC 19L was 73.6% after cultured in MRS broth medium with pH 3.0 for 2 h. The results showed that the strain CMRC 19L had good tolerance to acidic environment.
- the 16S rDNA of the strain was determined, and the sequencing primers were 27F (5'-AGAGTTTGATCMTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3').
- the 16S rDNA sequence result of the strain CMRC 19L is shown in SEQ ID No.1. It has more than 99% homology with the 16S rDNA sequence of Lactobacillus plantarum SK151.
- Lactobacillus plantarum (Lactobacillus plantarum) CMRC 19L has been preserved on September 9, 2015 in the General Microbiology Center of China Committee for Culture Collection of Microorganisms (CGMCC for short, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Microbiology, Chinese Academy of Sciences Research Institute, Zip Code 100101), the classification name is Lactobacillus plantarum, and the preservation number is CGMCC No.11347.
- Lactobacillus plantarum CGMCC No.11347, CGMCC No.9741, CGMCC No.9742, CGMCC No.11348 were respectively in containing 0.1%, 0.15%, 0.2%, 0.25%, 0.3% (W/V) sodium taurocholate After being successively subcultured in MRS broth medium, they were respectively inoculated on MRS agar medium containing 0.1%-0.3% (W/V) sodium taurocholate and MRS agar medium without sodium taurocholate, cultured at 37°C for 48 hours, and counted colonies. Calculate the survival rate of the strain.
- Lactobacillus plantarum CGMCC No.11347 The growth of Lactobacillus plantarum CGMCC No.11347, CGMCC No.9741, CGMCC No.9742, and CGMCC No.11348 on MRS agar medium containing different bile salt concentrations is shown in Table 1. Among them, Lactobacillus plantarum CGMCC No. 11347 had the highest survival rate under the conditions of 0.1%, 0.2%, and 0.3% (W/V) bile salt concentrations, indicating that it has good tolerance to bile salts.
- Lactobacillus plantarum CGMCC No.11347 has a strong inhibitory effect on Escherichia coli O157:H7 and Staphylococcus aureus.
- Lactobacillus Adhesion rate (%) CGMCC No.9741 8.43 CGMCC No.9742 0.04 CGMCC No.11347 7.53 CGMCC No.11348 5.22
- HT-29 cells were digested with trypsin solution, the cell concentration was adjusted to 5 ⁇ 10 5 /mL with DMEM nutrient solution, distributed in tissue culture plates, and incubated at 37°C for 24 hours. After washing the plate twice with sterile PBS buffer, mix 10 8 CFU/mL Lactobacillus plantarum CGMCC No.11347 with 1 mL of DMEM nutrient solution, add the mixed solution to the above tissue culture plate containing cells, and dilute at 5% Incubate at 37°C for 1.5h in a CO 2 -95% air environment.
- Lactobacillus The number of adhered bacteria (pcs/cell) CGMCC No.9741 12.2 CGMCC No.9742 3.2 CGMCC No.11347 16.7 CGMCC No.11348 11.3
- Inoculate Lactobacillus plantarum CGMCC No.11347 in MRS broth medium centrifuge after cultivating at 37°C for 16h, thalline and equal mass of lyoprotectant (per 100ml mass concentration is 10% skim milk with 2g Glycerin and 3g sucrose) were mixed, pre-frozen at -20°C for 2h, then placed in a vacuum freeze dryer for vacuum drying (cold trap temperature -45°C, vacuum degree 10-20Pa) for 22h-24h, and the water content of the bacteria powder was reduced to 2.5%. Stop drying at -3% to obtain a dry powder inoculum, the content of live Lactobacillus plantarum in the inoculum is 10 11 CFU/g.
- the fermented aroma is the strongest and there is no unpleasant smell as 7 points, and the lighter the fermented aroma or unpleasant smell is, the lower the score is; the stipulated taste has a pleasant fermented taste and suitable acidity is 5 points The lighter the fermentation taste, the lower the score.
- the evaluation of the results is mainly through the olfactory sensory evaluation of people of different genders and ages. There are 12 people in each group participating in the odor evaluation, the ratio of male to female is 1:1, and the age is between 25 and 40.
- the low-salt fermented sausage prepared by the method of the present embodiment the addition of sodium chloride is 2%, and the production cycle is reduced from more than 50 days to about 20 days; in the product, harmful bacteria such as Escherichia coli O157:H7, Staphylococcus aureus, etc. Not detected; the mean scores of odor, taste and overall acceptability of fermented sausages added Lactobacillus plantarum CGMCC No.11347 were higher than those of sausages without starter (Table 5).
- SPF grade SD rats clean grade, half male and half male, body weight (200 ⁇ 20) g, same age.
- Males and females were reared in separate cages at a room temperature of 25-28°C and a humidity of about 70%. They were fed regularly every day, weighed once a week and recorded their food intake. After 2-3 days of feeding for rat adaptability, the unqualified rats were eliminated, and the rats were randomly divided into control group (basic feed, group 1), fresh pork group (fresh pork, group 2), fermented sausage group (the fermented sausage that above-mentioned embodiment 4 makes, the 3rd group), totally 3 experimental groups. 16-20 rats in each group, half male and half male.
- each group was rationed with basal feed, fresh pork and fermented meat.
- the fresh pork and the fermented meat are respectively added into sterile physiological saline and homogeneously mixed to prepare a suspension.
- Diet Pagoda based on an adult body weight of 60kg, the basic body weight of experimental rats is calculated on the basis of 200g per rat, and the rats eat 200mg of fresh pork and fermented meat per rat.
- Daily regular gavage 1 continuous 60 days.
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Abstract
提供了一株益生植物乳杆菌CMRC 19L及其在低盐发酵肉食品制备中的应用,该植物乳杆菌CMRC 19L保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏编号为CGMCC No.11347。植物乳杆菌CMRC 19L的生长速度快、可抑制病原菌生长,具有优良的肉类发酵性能,同时耐受胃肠道环境,对肠黏液蛋白及肠上皮样细胞均表现出较高的黏附性,具有良好的益生性能。
Description
本申请要求于2022年02月24日提交中国专利局、申请号为CN202210169296.4、发明名称为“一株益生植物乳杆菌及其在低盐发酵肉食品制备中的应用”的中国专利申请的优先权,其全部内容通过引用结合在本申请中。
本发明涉及微生物技术领域,具体涉及一株益生植物乳杆菌及其在低盐发酵肉食品制备中的应用。
发酵肉食品具有保质期长,风味独特的特点,深受消费者喜爱。为了保证食用安全性,发酵肉生产中的氯化钠添加量通常高达4-10%。较高的氯化钠含量容易引起消费者高血压和心血管等疾病,不利于人体健康。随着人们对健康的日益关注,可改善肠道菌群、促进人体健康的低盐发酵肉食品受到越来越多消费者的青睐。
乳酸菌对低盐发酵肉食品的安全、风味和营养品质具有重要影响。研究表明,乳酸菌能通过产酸降低pH值和产细菌素抑制低盐发酵肉中有害菌生长,保证低盐发酵肉食品的微生物安全性;乳酸菌可以代谢碳水化合物、肌肉蛋白和脂肪,产生酸类、酯类、肽等风味物质,从而提高低盐发酵肉食品的感官风味品质。近年来,可用于低盐发酵肉食品的益生乳酸菌的研究与应用,也越来越受到关注。
目前,来源于人体肠道的益生乳酸菌研究报道较多,这是由于这些乳酸菌更可能具有耐胃酸、高肠道黏附能力等益生性能。然而,与来源于自然发酵肉的乳酸菌相比,来源于人体肠道的乳酸菌的肉类发酵性能往往并不理想,不适合作为肉类发酵剂。目前兼具优良肉类发酵性能和益生性能的乳酸菌发酵剂少见报道。此外,与多菌株复配发酵剂相比,兼具优良肉类发酵性能和益生性能的单一菌株作为发酵剂,更有利于降低生产成本。
中国专利CN113957021A公开了一种具有耐消化应激及高肠道黏附能力的植物乳杆菌grx401及其应用,该菌株具有耐消化应激及高肠道黏附能力,但来源于人体肠道,并且未报道该菌株具有优良的肉类发酵性能。中国专利 CN108410774B公开了一种复配菌剂及其在功能发酵肉食品中的应用,其使用的是包含植物乳杆菌和木糖葡萄球菌的复配菌剂。中国专利CN113373097A公开了一株植物乳杆菌及其针对现有川味香肠发酵中的应用,但并未报道该菌株具有优良的益生性能,且使用的是植物乳杆菌、戊糖片球菌和葡萄球菌的复配菌剂。
发明内容
本发明的目的是提供一株益生植物乳杆菌及其在低盐发酵肉食品制备中的应用。
具体地,本发明提供以下技术方案:
本发明提供植物乳杆菌(Lactobacillus plantarum)CMRC 19L,该菌株于2015年9月9日保藏于中国微生物菌种保藏管理委员会普通微生物中心(简称CGMCC,地址:北京市朝阳区北辰西路1号院3号,中国科学院微生物研究所,邮编100101),分类命名为植物乳杆菌Lactobacillus plantarum,保藏编号为CGMCC No.11347。
植物乳杆菌CMRC 19L的16S rDNA基因序列如SEQ ID No.1所示。
本发明提供一种发酵物,其由植物乳杆菌CMRC 19L经发酵培养制备得到。所述发酵物包含植物乳杆菌CMRC 19L或包含植物乳杆菌CMRC 19L及其发酵上清液。
本发明提供一种菌剂,其含有植物乳杆菌CMRC 19L。
本发明还提供一种发酵剂,其含有植物乳杆菌CMRC 19L。
优选地,上述菌剂、发酵剂中植物乳杆菌CMRC 19L以活菌形式存在。
优选地,上述菌剂、发酵剂还含有冻干保护剂。
上述菌剂或发酵剂可以为液体菌剂、发酵剂或固体菌剂、发酵剂(例如:干粉菌剂)。
本发明提供所述菌剂或所述发酵剂的制备方法,所述方法包括将植物乳杆菌CMRC19L经培养获得菌液的步骤。
优选地,所述培养为在37℃条件下进行。培养使用的培养基包括但不限于MRS肉汤培养基等植物乳杆菌常用的培养基。
上述获得的菌液可直接或加入微生物制剂领域允许的辅料制备为液体菌剂或发酵剂,也可收集菌液中的菌体与冻干保护剂等微生物制剂领域允许的辅料 混合后经真空冷冻干燥制备为干粉菌剂或发酵剂。
优选地,所述菌剂或发酵剂中,植物乳杆菌CMRC 19L的活菌含量为10
10-10
12CFU/g。
本发明通过实验证明,植物乳杆菌CMRC 19L的生长速度快,对于致病性大肠杆菌、金黄色葡萄球菌等病原菌具有明显的抑制作用,不仅具有优良的肉类发酵性能,能够较好地保证发酵肉食品的食用安全性和风味品质,而且,能够耐受胃肠道环境,且对于肠黏液蛋白和肠上皮细胞均具有较高的黏附性,具有优异的益生性能。
基于植物乳杆菌CMRC 19L的功能,本发明提供植物乳杆菌CMRC 19L或所述菌剂或所述发酵剂在发酵食品制备中的应用;
以及,植物乳杆菌CMRC 19L或所述菌剂或所述发酵剂在制备用于抑制病原微生物、补充肠道益生菌或控制体重增长的产品中的应用。
上述应用中,所述发酵食品优选为发酵肉食品;更优选为低盐发酵肉食品;最优选为低盐发酵香肠。
上述应用中,所述病原微生物优选为致病性大肠杆菌或金黄色葡萄球菌。
上述应用中,所述产品包括但不限于食品、饲料、益生菌制剂或药物。
本发明提供一种低盐发酵肉食品,所述低盐发酵肉食品为将包含畜禽肉的原料利用植物乳杆菌CMRC 19L或所述菌剂或所述发酵剂进行发酵制备得到。
优选地,所述发酵的接种量为10
9-10
11CFU植物乳杆菌CMRC 19L/kg原料。更优选为10
10CFU植物乳杆菌CMRC 19L/kg原料。
所述发酵为先于28-32℃、湿度90±3%的条件下发酵1-3天,再于13-17℃、湿度80±3%的条件下发酵16-20天。
优选地,所述发酵为先于30℃、湿度90±3%的条件下发酵2天,再于15℃、湿度80±3%的条件下发酵18天。
所述原料包含畜禽肉以及占畜禽肉质量百分比为1-3%的氯化钠。
优选地,氯化钠占畜禽肉的质量百分比为1-2%。
所述畜禽肉包括但不限于猪肉、羊肉、鸡肉或牛肉,可包括瘦肉和/或脂肪。
优选地,所述原料还包括香辛料和/或添加剂。
作为本发明的优选方案,所述原料包括如下组分:瘦肉750-850g,脂肪150-250g,氯化钠10-20g,黑胡椒1-3g,白胡椒1-3g,葡萄糖5-15g,亚硝酸 钠0.05-0.1g,异VC钠0.1-1g,三聚磷酸盐1-3g,干红葡萄酒5-15g,鲜蒜3-6g,水15-25g。
本发明还提供一种低盐发酵肉食品的制备方法,所述方法包括将包含畜禽肉的原料利用植物乳杆菌CMRC 19L或所述菌剂或所述发酵剂进行发酵的步骤。
优选地,所述发酵的接种量为10
9-10
11CFU植物乳杆菌CMRC 19L/kg原料。更优选为10
10CFU植物乳杆菌CMRC 19L/kg原料。
所述发酵为先于28-32℃、湿度90±3%的条件下发酵1-3天,再于13-17℃、湿度80±3%的条件下发酵16-20天。
优选地,所述发酵为先于30℃、湿度90±3%的条件下发酵2天,再于15℃、湿度80±3%的条件下发酵18天。
所述原料包含畜禽肉以及占畜禽肉质量百分比为1-3%的氯化钠。
优选地,氯化钠占畜禽肉的质量百分比为1-2%。
所述畜禽肉包括但不限于猪肉、羊肉、鸡肉或牛肉,可包括瘦肉和/或脂肪。
优选地,所述原料还包含香辛料和/或添加剂。
作为本发明的优选方案,所述原料包括如下组分:瘦肉750-850g,脂肪150-250g,氯化钠10-20g,黑胡椒1-3g,白胡椒1-3g,葡萄糖5-15g,亚硝酸钠0.05-0.1g,异VC钠0.1-1g,三聚磷酸盐1-3g,干红葡萄酒5-15g,鲜蒜3-6g,水15-25g。
具体地,所述方法包括如下步骤:
(1)将新鲜猪肉和脂肪切割、斩拌后,将瘦肉与脂肪颗粒混合均匀;
(2)将混合均匀的瘦肉与脂肪颗粒与氯化钠、香辛料和添加剂混合,得到原料;
(3)将植物乳杆菌CMRC 19L按照每千克原料中添加10
9-10
11CFU的比例接种后,灌制香肠;
(4)将灌制的香肠先在30℃、湿度90±3%的条件下发酵2天,再于15℃、湿度80±3%的条件下发酵18天使其成熟。
本发明的有益效果在于:本发明提供一株植物乳杆菌CMRC 19L,该菌株从传统自然发酵酸肉中分离获得,生长速度快、可抑制病原菌生长、具有优良的肉类发酵性能,同时耐受胃肠道环境,对肠黏液蛋白及肠上皮样细胞均表现 出较高黏附性,具有良好的益生性能。该菌株作为发酵剂可保证低盐发酵肉食品的食用安全性和风味品质,缩短发酵周期,并显著提高发酵肉食品的营养价值。
图1为本发明实施例2中植物乳杆菌CGMCC No.11347培养上清液对大肠杆菌O157:H7和金黄色葡萄球菌的抑制效果,其中,对比菌株CGMCC No.9741、CGMCC No.9742、CGMCCNo.11348均为植物乳杆菌,都分离自贵州传统自然发酵酸肉,左图为金黄色葡萄球菌的抑制效果,右图为大肠杆菌O157:H7的抑制效果;
图2为本发明实施例2中植物乳杆菌CGMCC No.11347的黏附细胞图(400×);
图3为本发明实验例中大鼠肠道乳酸菌活菌数的检测结果,分别饲喂基础饲料(空白对照)、新鲜猪肉、发酵香肠连续30天和60天后,雌雄大鼠肠道乳酸菌活菌数;
图4为本发明实验例中大鼠体重增加的检测结果,分别饲喂基础饲料(空白对照)、新鲜猪肉、发酵香肠连续30天和60天后,雌雄大鼠增加的体重;
图3和图4中,“雄性-30天”为雄性大鼠连续饲喂30天、“雌性-30天”为雌性大鼠连续饲喂30天、“雄性-60天”为雄性大鼠连续饲喂60天、“雌性-60天”为雌性大鼠连续饲喂60天。
生物保藏说明
植物乳杆菌(Lactobacillus plantarum)CMRC 19L,于2015年9月9日保藏于中国微生物菌种保藏管理委员会普通微生物中心(简称CGMCC),保藏地址:北京市朝阳区北辰西路1号院3号,中国科学院微生物研究所,邮编100101),保藏编号为CGMCC No.11347。
以下实施例用于说明本发明,但不用来限制本发明的范围。
在本发明中,若非特指,所有的份、百分比均为重量单位,所采用的设备和原料等均可从市场购得或是本领域常用的。以下实施例中的方法,如无特别说明,均为本领域的常规方法。
以下实施例中涉及的对照菌株CGMCC No.9741已于2014年9月28日保 藏于中国微生物菌种保藏管理委员会普通微生物中心(简称为CGMCC,地址为北京市朝阳区北辰西路1号院3号,中国科学院微生物研究所,邮政编码:100101)保藏编号为CGMCC No.9741,该菌株已在专利申请CN 104560808 A中公开。
CGMCC No.9742已于2014年9月28日保藏于中国微生物菌种保藏管理委员会普通微生物中心,其简称为CGMCC,地址为北京市朝阳区北辰西路1号院3号中国科学院微生物研究所,邮政编码:100101,保藏编号为CGMCCNo.9742,该菌株已在专利申请CN 104560802A中公开。
CGMCC No.11348已于2015年09月09日保藏于中国微生物菌种保藏管理委员会普通微生物中心,其简称为CGMCC,地址为北京市朝阳区北辰西路1号院3号中国科学院微生物研究所,邮政编码:100101,保藏编号为CGMCC No.11348。该菌株已在专利申请CN108410774A中公开。
挥发性风味物质含量检测参照K.A.Gutsche,T.B.Tran,R.F.Vogel,Production of volatile compounds by Lactobacillus sakei from branched chainalpha-keto acids[J].Food Microbiology,2012,29:224-228.中所提供的方法(SPME-GC-MS法)。
游离氨基酸含量检测参照李平兰,沈清武,等.微生物酶与肉组织酶对干发酵香肠中游离氨基酸的影响[J].食品与发酵工业,2005,31(5):134-138.中所提供的方法。
实施例1植物乳杆菌CMRC 19L的分离和鉴定
1.1菌株分离和纯化
本发明提供的植物乳杆菌CMRC 19L分离自贵州传统自然发酵酸肉:取25g酸肉样品,加入225mL灭菌生理盐水中,拍打均质8min。取上清液,梯度稀释后涂布于MRS琼脂培养基上,在30℃培养48h后,从最高稀释度的平板上随机挑取边缘整齐、光滑的单菌落进一步培养纯化。
将得到的菌株进行形态学、耐盐性、耐亚硝酸盐性、葡萄糖同型/异型发酵等理化测定,选择革兰氏染色镜检为阳性、耐受8%氯化钠和0.015%亚硝酸钠、同型或兼性异型发酵的菌株CMRC 19L,进行进一步鉴定。
1.2菌株耐酸性检测
将活菌数量为3×10
8/mL的菌株CMRC 19L菌悬液,按5%(V/V)的比 例接种于pH3.0的MRS肉汤培养基中,37℃厌氧培养0h、2h后,进行平板计数计算菌株存活率。
在pH为3.0的MRS肉汤培养基中培养2h后,菌株CMRC 19L的存活率为73.6%。结果表明,菌株CMRC 19L对酸性环境的耐受性良好。
1.3菌株鉴定
对菌株的16S rDNA进行测定,测序引物为27F(5’-AGAGTTTGATCMTGGCTCAG-3’)和1492R(5’-GGTTACCTTGTTACGACTT-3’),菌株CMRC 19L的16S rDNA序列结果如SEQ ID No.1所示,与植物乳杆菌SK151的16S rDNA序列同源性超过99%。
植物乳杆菌(Lactobacillusplantarum)CMRC 19L已于2015年9月9日保藏于中国微生物菌种保藏管理委员会普通微生物中心(简称CGMCC,地址:北京市朝阳区北辰西路1号院3号,中国科学院微生物研究所,邮编100101),分类命名为植物乳杆菌Lactobacillus plantarum,保藏编号为CGMCC No.11347。
实施例2植物乳杆菌CGMCC No.11347的益生性能检测
2.1胆盐耐受性试验
将植物乳杆菌CGMCC No.11347、CGMCC No.9741、CGMCC No.9742、CGMCC No.11348分别在含有0.1%、0.15%、0.2%、0.25%、0.3%(W/V)牛胆酸钠的MRS肉汤培养基中依次传代后,分别接种于含有0.1%-0.3%(W/V)牛胆酸钠和不含牛胆酸钠的MRS琼脂培养基,37℃培养48h后进行菌落计数,计算菌株存活率。
植物乳杆菌CGMCC No.11347、CGMCC No.9741、CGMCC No.9742、CGMCC No.11348分别在含不同胆盐浓度的MRS琼脂培养基上的生长情况见表1。其中,植物乳杆菌CGMCCNo.11347在0.1%、0.2%、0.3%(W/V)胆盐浓度条件下存活率均为最高,说明其对胆盐的耐受性良好。
2.2模拟肠液的耐受性试验
将牛胆盐酸钠(0.2%)和胰蛋白酶(1%)溶解于10mL无菌生理盐水中,pH值调节至8.0制备模拟肠液。将活菌数量为3×10
8/mL的植物乳杆菌CGMCC No.11347、CGMCCNo.9741、CGMCC No.9742、CGMCC No.11348的菌悬液,按1%(V/V)的比例接种于模拟肠液中,37℃厌氧培养0h、2h后,进行平板计数计算菌株存活率。
结果如表1所示,植物乳杆菌CGMCC No.11347在模拟肠液中处理2h后活菌数最高,说明该菌株能够耐受模拟肠道环境,具备良好的肠道生存能力。
表1植物乳杆菌CGMCC No.11347对模拟肠道环境的耐受性能
2.3病原菌抑制试验
将植物乳杆菌CGMCC No.11347、CGMCC No.9741、CGMCC No.9742、CGMCC No.11348分别接种于MRS肉汤培养基,37℃培养24h后,4000rpm离心10min,上清液用0.22μm微孔滤膜过滤。将大肠杆菌O157:H7和金黄色葡萄球菌分别接种于营养肉汤中,37℃培养24h后,4000rpm离心10min,用无菌生理盐水制成活菌数为3.0×10
8/mL的菌悬液。无菌平皿中加入2%无菌琼脂10mL,琼脂凝固后放入无菌牛津杯
将200μL大肠杆菌O157:H7或金黄色葡萄球菌菌悬液与15mL营养琼脂混匀倒入平皿,琼脂凝固后取出牛津杯。在孔中加入100μL植物乳杆菌CGMCC No.11347、CGMCC No.9741、CGMCC No.9742或CGMCC No.11348的上清液,37℃培养20h后观察有无抑菌圈。
结果如图1及表2所示,植物乳杆菌CGMCC No.11347对大肠杆菌O157:H7和金黄色葡萄球菌具有较强的抑制作用。
表2植物乳杆菌CGMCC No.11347抑菌试验结果
注:-代表无抑菌圈。
2.4黏附能力
2.4.1对肠黏液蛋白的黏附能力
在96孔板的每孔加入200μL猪肠黏液蛋白溶液,过夜固定后去除未固定的黏液。每孔加入活菌数量为3.0×10
8/mL的植物乳杆菌CGMCC No.11347菌悬液100μL,37℃孵育1h后用PBS缓冲液(pH 4.0)清洗除去未黏附的植物乳杆菌。将96孔板置于60℃烘干30min后,每孔加入100μL 0.1%结晶紫水溶液染色50min,用PBS缓冲液清洗后加入100μL柠檬酸盐缓冲液,室温孵育60min后,测定540nm处吸光值。以黏附乳酸菌数占加入乳酸菌数的百分比表示菌株的黏附能力。
结果如表3显示,植物乳杆菌CGMCC No.11347对肠黏液蛋白的黏附率为7.53%,具有一定的肠黏液蛋白黏附能力。植物乳杆菌CGMCC No.11347和CGMCC No.9741的肠黏液蛋白黏附能力差异不显著。
表3植物乳杆菌CGMCC No.11347对肠黏液蛋白的黏附能力
乳杆菌 | 黏附率(%) |
CGMCC No.9741 | 8.43 |
CGMCC No.9742 | 0.04 |
CGMCC No.11347 | 7.53 |
CGMCC No.11348 | 5.22 |
2.4.2对人肠上皮样细胞HT-29的黏附能力
将HT-29细胞用胰酶消化液进行消化后,利用DMEM营养液调整细胞浓度为5×10
5/mL并分装于组织培养板中,37℃培养24h。用无菌PBS缓冲液洗板2次后,将10
8CFU/mL植物乳杆菌CGMCC No.11347与1mL的DMEM营 养液混合,将混合液加入上述装有细胞的组织培养板中,于5%CO
2-95%空气环境中37℃培养1.5h。无菌PBS缓冲液洗板5次后,用8%甲醛溶液固定2h,再用无菌PBS缓冲液洗板3次。革兰氏染色后于显微镜下观察,随机选取20个视野记录黏附的总菌数和总细胞数,其比值即为每个细胞上黏附的细菌数。
表4植物乳杆菌CGMCC No.11347对人肠上皮样细胞HT-29的黏附能力
乳杆菌 | 黏附的细菌数(个/cell) |
CGMCC No.9741 | 12.2 |
CGMCC No.9742 | 3.2 |
CGMCC No.11347 | 16.7 |
CGMCC No.11348 | 11.3 |
结果如图2和表4所示,植物乳杆菌CGMCC No.11347对肠上皮样细胞HT-29的黏附率为16.7CFU/细胞,表明该菌具有良好的细胞黏附能力,能够黏附于人肠道定植以发挥作用。
实施例3发酵剂菌粉的制备
将植物乳杆菌CGMCC No.11347接种于MRS肉汤培养基中,37℃培养16h后离心,将菌体与等质量的冻干保护剂(每100ml质量浓度为10%的脱脂乳中添加有2g甘油和3g蔗糖)混合,-20℃预冻2h后,置于真空冷冻干燥机中真空干燥(冷阱温度-45℃,真空度10-20Pa)22h-24h,菌粉含水量降至2.5%-3%时停止干燥,即得到干粉形式的菌剂,菌剂中植物乳杆菌活菌含量为10
11CFU/g。
实施例4低盐发酵肉食品的生产
将新鲜猪肉和脂肪切成边长为2-3cm的立方体经冷冻后进行斩拌,将瘦肉与脂肪颗粒混合均匀后,加入香辛料和添加剂(猪肉800g,猪背脂肪200g,氯化钠20g,黑胡椒2g,白胡椒2g,葡萄糖10g,亚硝酸钠0.1g,异VC钠0.5g,三聚磷酸盐3g,干红葡萄酒12g,鲜蒜5g,水20g)。将实施例3中制备的菌剂按每千克肉馅中添加0.1g的比例加入肉馅中。将灌制的香肠在30℃、湿度90±3%的条件下发酵2天,15℃、湿度80±3%的条件下发酵18天使其成熟。
感官风味评价:规定发酵香气最浓且没有令人不愉快的气味为7分,发酵香气越淡或有不愉快的气味则分值越低;规定滋味具有令人愉悦的发酵味道且酸度适宜的为5分,发酵味道越淡则分值越低。对结果的评定主要通过不同性别、不同年龄段的人进行嗅觉感官评定。各组参与气味评价的人数为12人,男 女比例均为1:1,年龄在25~40之间。
本实施例的方法制得的低盐发酵香肠,氯化钠添加量2%,生产周期从通常50天以上降低到20天左右;产品中大肠杆菌O157:H7、金黄色葡萄球菌等有害菌均未检出;添加植物乳杆菌CGMCC No.11347的发酵香肠气味、滋味和总体可接受性的平均分均高于未添加发酵剂的香肠(表5)。
表5植物乳杆菌CGMCC No.11347生产发酵香肠的感官风味评分
本实施例制备的低盐发酵香肠的挥发性风味物质的检测结果如表6所示。与未添加发酵剂的发酵香肠相比,植物乳杆菌发酵剂的添加使发酵香肠中挥发性风味物质种类略有增加,同时将发酵香肠中挥发性风味物质的总含量提高了近1倍。
表6植物乳杆菌CGMCC No.11347生产发酵香肠的风味物质种类和含量
本实施例制备的低盐发酵香肠的游离氨基酸的检测结果如表7所示。与未添加发酵剂的发酵香肠相比,植物乳杆菌发酵剂的添加将发酵香肠中游离氨基酸总含量提高了20%。
表7植物乳杆菌CGMCC No.11347生产发酵香肠的游离氨基酸含量
实验例动物实验
SPF级SD大鼠,清洁级,雌雄各半,体质量(200±20)g,日龄一致。雌雄分笼饲养,室温25-28℃,湿度70%左右,每天定时饲喂,每周称量1次体质量并记录进食量。大鼠适应性观察喂养2-3天后,剔除不合格大鼠,大鼠随机数字法分为对照组(基础饲料,第1组)、新鲜猪肉组(新鲜猪肉,第2组)、发酵香肠组(上述实施例4制得的发酵香肠,第3组),共计3个实验组。每组16-20只大鼠,雌雄各半。正式实验起,各组定量给予基础饲料、新鲜猪肉和发酵肉。将新鲜猪肉和发酵肉分别加入无菌生理盐水均质混匀,制成悬浮液。参考中国居民膳食宝塔对畜禽肉类食物每日推荐量,以成年人体质量60kg计,实验大鼠基础体质量以每只200g计,大鼠每日摄食新鲜猪肉和发酵肉200mg/只,每日定时灌胃1次,连续60天。
实验30天和60天时,记录对比各实验组大鼠肠道乳酸菌活菌数。如图3所示,连续饲喂发酵香肠提高了雌雄大鼠肠道乳酸菌的活菌数量,其中30天时发酵香肠组雌雄大鼠的肠道乳酸菌数量显著高于新鲜猪肉组,60天时发酵香肠组雌性大鼠的肠道乳酸菌数量显著高于新鲜猪肉组(*表示差异显著,P<0.05)。
实验30天和60天时,记录对比各实验组大鼠体重。如图4所示,饲喂新鲜猪肉可导致不同性别大鼠体质量显著增加,而饲喂发酵香肠可显著抑制雄、雌性大鼠体质量增加(*表示差异显著,P<0.05)。
虽然,上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述,但在本发明基础上,可以对之作一些修改或改进,这对本领域技术人员而言是显而易见的。因此,在不偏离本发明精神的基础上所做的这些修改或改进,均属于本发明要求保护的范围。
Claims (23)
- 植物乳杆菌(Lactobacillusplantarum)CMRC 19L,其特征在于,所述植物乳杆菌CMRC 19L保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏编号为CGMCC No.11347。
- 一种菌剂,其特征在于,所述菌剂含有权利要求1所述的植物乳杆菌CMRC 19L。
- 根据权利要求2所述的菌剂,其特征在于,所述菌剂为一种发酵剂。
- 根据权利要求2或3所述的菌剂,其特征在于,所述菌剂中所述植物乳杆菌CMRC 19L的活菌含量为10 10-10 12CFU/g。
- 根据权利要求2或3所述的菌剂,其特征在于,所述菌剂还含有冻干保护剂。
- 根据权利要求2或3所述的菌剂,其特征在于,所述菌剂为液体菌剂或固体菌剂。
- 权利要求2~6任一项所述的菌剂的制备方法,其特征在于,包括将植物乳杆菌CMRC 19L经培养获得菌液的步骤。
- 根据权利要求7所述的制备方法,其特征在于,所述培养的温度为37℃;所述培养使用的培养基包括MRS肉汤培养基。
- 权利要求1所述的植物乳杆菌CMRC 19L或权利要求2~6任一项所述的菌剂在发酵食品制备中的应用。
- 根据权利要求9所述的应用,其特征在于,所述发酵食品为发酵肉食品。
- 根据权利要求10所述的应用,其特征在于,所述发酵肉食品为低盐发酵肉食品。
- 权利要求1所述的植物乳杆菌CMRC 19L或权利要求2~6任一项所述的菌剂在制备用于抑制病原微生物、补充肠道益生菌或控制体重增长的产品中的应用。
- 根据权利要求12所述的应用,其特征在于,所述病原微生物包括致病性大肠杆菌或金黄色葡萄球菌。
- 根据权利要求12或13所述的应用,其特征在于,所述产品包括食品、饲料、益生菌制剂或药物。
- 一种低盐发酵肉食品,其特征在于,所述低盐发酵肉食品为将包含畜禽 肉的原料利用权利要求1所述的植物乳杆菌CMRC 19L或权利要求2~6任一项所述的菌剂进行发酵得到。
- 根据权利要求15所述的低盐发酵肉食品,其特征在于,所述发酵的接种量为10 9-10 11CFU植物乳杆菌CMRC 19L/kg原料。
- 根据权利要求15或16所述的低盐发酵肉食品,其特征在于,所述发酵为先于28-32℃、湿度90±3%的条件下发酵1-3天,再于13-17℃、湿度80±3%的条件下发酵16-20天。
- 根据权利要求15或16所述的低盐发酵肉食品,其特征在于,所述原料包含畜禽肉以及占畜禽肉质量百分比为1-3%的氯化钠。
- 根据权利要求18所述的低盐发酵肉食品,其特征在于,所述畜禽肉包括猪肉、羊肉、鸡肉或牛肉。
- 根据权利要求19所述的低盐发酵肉食品,其特征在于,所述畜禽肉包括瘦肉和/或脂肪。
- 根据权利要求16所述的低盐发酵肉食品,其特征在于,所述原料还包括香辛料和/或添加剂。
- 根据权利要求16所述的低盐发酵肉食品,其特征在于,所述原料包括如下组分:瘦肉750-850g,脂肪150-250g,氯化钠10-20g,黑胡椒1-3g,白胡椒1-3g,葡萄糖5-15g,亚硝酸钠0.05-0.1g,异VC钠0.1-1g,三聚磷酸盐1-3g,干红葡萄酒5-15g,鲜蒜3-6g,水15-25g。
- 根据权利要求22所述的低盐发酵肉食品,其特征在于,所述低盐发酵肉食品的制备方法包括:将新鲜猪肉和脂肪切割、斩拌后,将瘦肉与脂肪颗粒混合均匀;将所述混合均匀的瘦肉与脂肪颗粒与氯化钠、香辛料和添加剂混合,得到原料;将植物乳杆菌CMRC 19L按照每千克所述原料中添加10 9-10 11CFU的比例接种后,灌制香肠;将灌制的所述香肠先在30℃、湿度90±3%的条件下发酵2天,再于15℃、湿度80±3%的条件下发酵18天使其成熟。
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CN114591872B (zh) * | 2022-04-12 | 2023-04-25 | 东北农业大学 | 一种风干肠发酵剂及其应用以及风干肠的制备方法 |
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