CN111690573A - Lactobacillus hilgardii for animal intestinal probiotics, ferment prepared by fermentation of lactobacillus hilgardii and application of ferment - Google Patents
Lactobacillus hilgardii for animal intestinal probiotics, ferment prepared by fermentation of lactobacillus hilgardii and application of ferment Download PDFInfo
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- CN111690573A CN111690573A CN202010710659.1A CN202010710659A CN111690573A CN 111690573 A CN111690573 A CN 111690573A CN 202010710659 A CN202010710659 A CN 202010710659A CN 111690573 A CN111690573 A CN 111690573A
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- fermentation
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- lactobacillus hilgardii
- molasses
- ferment
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Classifications
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- 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
- C12N1/205—Bacterial isolates
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- 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
- A23K10/12—Animal feeding-stuffs obtained by microbiological or biochemical processes by fermentation of natural products, e.g. of vegetable material, animal waste material or biomass
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/30—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/30—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms
- A23K10/33—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms from molasses
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/105—Aliphatic or alicyclic compounds
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/142—Amino acids; Derivatives thereof
- A23K20/147—Polymeric derivatives, e.g. peptides or proteins
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/163—Sugars; Polysaccharides
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/189—Enzymes
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/20—Inorganic substances, e.g. oligoelements
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/20—Inorganic substances, e.g. oligoelements
- A23K20/30—Oligoelements
-
- 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
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
- Y02P60/80—Food processing, e.g. use of renewable energies or variable speed drives in handling, conveying or stacking
- Y02P60/87—Re-use of by-products of food processing for fodder production
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Polymers & Plastics (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Food Science & Technology (AREA)
- Animal Husbandry (AREA)
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- Organic Chemistry (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biomedical Technology (AREA)
- Genetics & Genomics (AREA)
- Microbiology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Virology (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Tropical Medicine & Parasitology (AREA)
- Medicinal Chemistry (AREA)
- Sustainable Development (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention belongs to the field of microorganism and fermentation engineering, and particularly relates to lactobacillus hilgardii for animal intestinal probiotics, and ferment prepared by fermentation and application thereof. The lactobacillus hilgardii provided by the invention is separated from rotten pineapples, is more suitable for fermentation of fruit juice, and has better acid production performance in a culture medium taking fruit juice as a main raw material compared with the existing lactobacillus for feed fermentation. The lactic acid bacteria used in the present application are also capable of producing antibacterial peptides and organic acids. Under the synergistic action of the antibacterial peptide, the organic acid and the enzyme preparation, respective performances can be further exerted, so that the product can promote the growth of animals and enhance the immunity and disease resistance of the animals when being used in the fields of feed and cultivation. Meanwhile, the enzyme prepared by the strain has good stability, and the activity of the enzyme in the product is still kept more than 90% after the enzyme is stored for 6 months at room temperature.
Description
Technical Field
The invention belongs to the field of microorganism and fermentation engineering, and particularly relates to lactobacillus hilgardii for animal intestinal probiotics, and ferment prepared by fermentation and application thereof.
Background
The microbial ferment is a functional microbial fermentation product which is produced by fermenting one or more fresh vegetables, fruits, mushrooms, Chinese herbal medicines and the like serving as raw materials by using a plurality of beneficial bacteria and contains abundant nutritional ingredients such as vitamins, enzymes, minerals, secondary metabolites and the like. In recent years, a great deal of research on enzymes in the fields of feed and cultivation is promoted, in livestock and poultry production, the enzymes fed by the feed can improve the digestibility of the feed, reduce the disease incidence rate of digestive system diseases of livestock and poultry, enhance the immunity of livestock and poultry, catalyze and decompose the feed, improve the conversion rate and the utilization rate of the feed, improve the meat quality, improve the yield and the like, and meanwhile, the enzymes fed by the feed can also reduce the residual quantity of veterinary drugs in livestock and poultry bodies, reduce the odor of livestock and poultry excrement and the like. No. 194 bulletin issued by rural departments in agriculture requires that all varieties of growth-promoting drug feed additives except traditional Chinese medicines are quitted from 1 month and 1 day in 2020, and the use of antibiotics is about to be completely forbidden by the feed industry. Undoubtedly, a good development opportunity is brought to the application of the functional enzyme product with the functions of enhancing the immunity and disease resistance of animals in the field of cultivation.
In recent years, the research on feed enzymes in China is gradually increased. However, most of the functional components of the ferment products for cultivation in the market are plant-derived functional components, and the ferment products are lack of microorganism-derived functional components, and have unstable quality and insignificant application effect during storage, so that the ferment products are not applied in the cultivation industry on a large scale. The domestic research on the feed enzyme is mainly focused on plant raw materials from different sources, currently, domestic scholars have researched enzymes from plants such as dendrobium, pineapples, apples and pineapples, but deep research is not carried out on the most key strains in enzyme production, and the strains are conventional probiotics such as lactic acid bacteria and saccharomycetes and lack functional strains for producing antibacterial peptides. As a green substitute of feed antibiotics, the antibacterial peptide generally has the characteristics of heat resistance, wide antibacterial spectrum, good water solubility, difficult generation of drug resistance and the like, and has good application prospect in the feed industry. The fermentation of the antibacterial peptide-rich ferment prepared by the antibacterial peptide-producing strain will undoubtedly further improve the function of the ferment. Therefore, it is urgent for the feed enzyme industry to screen highly effective antibacterial peptide-producing fermentation strains and to improve the storage stability of the products.
Lactobacillus hilgardii was first discovered in 1936 in high-alcoholicity wine by Douglas and Klaus and was named Lactobacillus hilgardii (the original isolate was lost in 1949), ten of which were re-isolated in the wine prepared by Rixi Van.En and Howard Douglas et al, university of California, USA. The Lactobacillus hilgardii can be used in the brewing process of yellow wine and the production of health food r-aminobutyric acid in the food industry. In recent years, lactobacillus hilgardii is widely applied to agriculture, effectively prevents and treats soil-borne diseases, and solves the problems of continuous cropping, death, soil deterioration and the like. However, the use of lactobacillus hilgardii as a feed probiotic and for producing feed ferment by fermentation has not been reported.
Disclosure of Invention
Aiming at the problems of lack of functional components, poor stability and the like of the existing ferment products, the invention aims to provide a Lactobacillus hilgardii (Lactobacillus hilgardii) YH-1 suitable for fruit fermentation, wherein the preservation number of the strain is CCTCC NO: m2020203.
Another objective of the invention is to provide an application of Lactobacillus hilgardii YH-1 in preparing fruit ferment.
The last objective of the present invention is to provide the application of the fruit ferment in the preparation of animal feed additives.
In order to achieve the purpose, the invention adopts the following technical measures:
obtaining lactobacillus hilgardii YH-1:
the applicant separates lactic acid bacteria from rotten pineapple dregs, further screens bacterial strains with antibacterial effects on escherichia coli and staphylococcus aureus, and finally screens Lactobacillus hilgardii YH-1 capable of producing antibacterial peptide through an acid discharge experiment, a hydrogen peroxide removal experiment and a protease sensitivity experiment, wherein the bacterial strains are preserved in China center for type culture collection (GmbH university) in 6-11 th 2020 at the preservation address of CCTCC NO: m2020203, taxonomic nomenclature: lactobacillus hilgardii (Lactobacillus hilgardii) YH-1.
The culture property of the lactobacillus hilgardii YH-1 separated by the method is the same as that of the conventional lactobacillus hilgardii.
The application of Lactobacillus hilgardii YH-1 in preparing fruit ferment comprises fermenting a culture medium containing fruit juice by using the Lactobacillus hilgardii provided by the invention;
in the above-mentioned application, the fermentation medium involved in the application process is:
the mass ratio of the molasses to the water is 0.01-0.5: 1, calcium chloride accounting for 0.01 to 5 percent of the total mass of the molasses and the water, ferrous chloride accounting for 0.01 to 5 percent of the total mass of the molasses and the water, ammonium sulfate accounting for 0.01 to 5 percent of the total mass of the molasses and the water, dipotassium hydrogen phosphate accounting for 0.01 to 5 percent of the total mass of the molasses and the water, fruit juice accounting for 5 to 25 percent of the total mass of the molasses and the water, and natural pH.
In the above-mentioned application, preferably, when it is used for preparing the bromelain, the preparation process comprises:
(1) mixing molasses and water according to the ratio of 0.01-0.5: 1, adding calcium chloride accounting for 0.01 to 5 percent of the total mass of the molasses and the water, ferrous chloride accounting for 0.01 to 5 percent of the total mass of the molasses and the water, ammonium sulfate accounting for 0.01 to 5 percent of the total mass of the molasses and the water and dipotassium hydrogen phosphate accounting for 0.01 to 5 percent of the total mass of the molasses and the water into the mixture, sterilizing and cooling the mixture to room temperature after uniformly mixing, and adding fruit juice accounting for 5 to 25 percent of the total mass of the molasses and the water into the mixture to;
(2) inoculating Lactobacillus hilgardii (Lactobacillus hilgardii) YH-1 seed liquid according to the inoculation amount of 1-9%, and standing and culturing for 24-40 h at 35-38 ℃; the mass percentage of the inoculation amount in the step is calculated according to the total mass of the fermentation medium;
(3) adding 0.01-5% of protease, 0.01-5% of cellulase, 0.01-5% of xylanase, 0.01-5% of mannase, 0.01-5% of glucose oxidase, 0.01-5% of chitosan, 0.01-5% of soybean protein and 0.01-5% of calcium propionate into a culture medium after fermentation is finished, and uniformly mixing to obtain the microbial fertilizer; the mass percentage in the step is calculated according to the total mass of the culture medium after the fermentation is finished;
the fruit juice is one or a mixture of two or three of pineapple juice, mulberry juice or green orange juice.
In the above method, preferably, the method for preparing fruit juice comprises:
washing fruit (without peeling) with sterile water, adding sterile water, squeezing, and filtering; the mass ratio of the fruits to the water is 1: 0.05 to 1.
Compared with the prior art, the invention has the following advantages:
(1) the strain has good fermentation performance. Most of the existing ferment is a natural fermentation strain or a strain used in feed fermentation. The lactic acid bacteria used in the present application are separated from the rotten pineapple and are more suitable for the fermentation of the fruit juice. Compared with the existing lactobacillus for fermenting feed, the lactobacillus has better acid production performance in a culture medium taking fruit juice as a main raw material.
(2) The product has more functional components. Most of the existing ferment products are endogenous enzymes and other components in plant raw materials. In addition to supplementing part of the enzyme preparation to compensate for the deficiency of endogenous enzymes in the plant material, the lactic acid bacteria used in the present invention can also produce antibacterial peptides and organic acids. Under the synergistic action of the antibacterial peptide, the organic acid and the enzyme preparation, respective performances can be further exerted, so that the product can promote the growth of animals and enhance the immunity and disease resistance of the animals when being used in the fields of feed and cultivation.
(3) The product has good stability. The existing enzyme product has poor stability, and the activity loss of the enzyme in the enzyme product is almost exhausted after the enzyme product is stored for 3 months at room temperature. After the ferment-based lactobacillus culture prepared by the invention is stored for 6 months at room temperature, the activity of the enzyme in the product is still kept more than 90 percent.
(4) The ferment prepared by the strain is suitable for aquatic products, advocated by poultry and mammals, has wide applicability, can promote the growth of raised animals and enhance the disease resistance of the animals, obviously reduces the breeding cost, and obtains remarkable economic benefit.
Detailed Description
The technical schemes of the invention are conventional schemes in the field if not particularly stated; the reagents or materials, if not specifically mentioned, are commercially available.
Example 1:
acquisition of Lactobacillus hilgardii (Lactobacillus hilgardii) YH-1 and its physio-biochemical properties:
(1) obtaining of Lactobacillus hilgardii (Lactobacillus hilgardii) YH-1
1) Selecting a rotten pineapple sample, weighing 10g of the pineapple sample, placing the pineapple sample in a triangular flask containing 90mL of sterile normal saline and glass beads, and uniformly mixing for 30min by using a vortex mixer to obtain 10g of pineapple-1The sample bacterial suspension is diluted to 10 times by gradient of 10 times by using sterile physiological saline－5. Are respectively from 10-3、10-4And 10-50.1mL of diluent is sucked from the sample bacterial suspensions with three dilutions, and the sample bacterial suspensions are respectively coated in MRS solid culture medium. The cells were cultured at 37 ℃ for 48 hours. The single colonies growing on the plate were gram stained, and gram-positive strains were selected.
2) Inoculating gram-positive bacteria on an inclined plane of an MRS solid culture medium, culturing for 48h at 37 ℃, selecting a ring with an aseptic inoculating ring, coating the ring on a clean glass slide, dropwise adding 3-5% of hydrogen peroxide, observing whether bubbles exist or not, if the bubbles exist, catalase shows a positive reaction, if the bubbles do not exist, catalase shows a negative reaction, and selecting a strain with catalase showing a negative reaction.
3) Screening out the strains which are gram-positive and catalase-negative, and preliminarily identifying the strains as the strains suspected to be lactic acid bacteria. Inoculating lactobacillus suspected strain into MRS liquid culture medium, standing at 37 deg.C for 48 hr, centrifuging fermented bacteria liquid at 10000r/min for 10min, removing thallus, and collecting supernatant. Firstly, 2.0% of vegetarian agar which is cooled to about 50-60 ℃ is injected into a sterilization plate, after cooling and solidification, 4 sterile oxford cups are evenly placed in the plate with the vegetarian agar by using sterile tweezers, then culture medium containing indicator bacteria is poured into the plate, and the oxford cups are pulled out by using the tweezers after solidification. Marking on Oxford cup holes, adding 200 mu L of lactobacillus fermentation supernatant into the Oxford cup holes, immediately placing the Oxford cup holes in a refrigerator at 4 ℃ for diffusion for 4h, transferring the Oxford cup holes to a corresponding temperature for growth of indicator bacteria (staphylococcus aureus and escherichia coli), culturing for a certain time, and observing whether a bacteriostatic zone exists. Screening 20 strains of lactic acid bacteria with bacteriostatic ability.
4) Respectively inoculating 20 strains of lactic acid bacteria with bacteriostatic ability into an MRS liquid culture medium for culturing for 48h, centrifuging the fermented bacteria liquid for 10min at 10000r/min, removing bacteria, reserving supernatant, adjusting the standby fermentation liquid of the lactic acid bacteria to be detected to p H5.0.0 by using 1.0mol/LNaOH, and performing bacteriostatic experiments by using a double-layer Oxford cup punching method, wherein the results show that only the strain YH-1 has bacteriostatic activity and the rest strains have no bacteriostatic activity.
(2) YH-1 is a lactic acid bacterium for producing antibacterial peptide
1) Taking 2.0mL of the fermentation broth of the strain YH-1, adjusting the pH value to 7.0, adding 20mg of catalase, slightly shaking for dissolution, placing at 37 ℃ for 2h, adjusting the pH value to 5.0, adjusting the pH value of the fermentation broth of the strain YH-1 which is not treated by the catalase to 5.0 as a control, and performing a bacteriostatic experiment by using a double-layer Oxford cup method.
2) Taking 3 parts of fermentation liquor of the strain YH-1, adjusting the pH to 7.0, adding papain and bacillus subtilis neutral protease according to the amount of 1.0mg/mL of final concentration, adding the same volume of sterile water into a control group, reacting for 4 hours at 50 ℃, and adjusting the pH to 5.0. A double-layer Oxford cup punching method is used for carrying out an antibacterial experiment, and the antibacterial activity of the fermentation liquor before and after protease enzymolysis is compared.
The acid-removing, hydrogen peroxide-removing and protease-sensitive experiments of the fermentation broth of strain YH-1 are shown in the following table. As can be seen from the results shown in the following table, the bacteriostatic substance produced by strain YH-1 can eliminate acid and hydrogen peroxide, and is a protein or polypeptide substance sensitive to protease.
Experimental result of strain YH-1 acid discharge, hydrogen peroxide discharge and protease sensitivity
Strain YH-1 was rod-shaped, approximately 0.5 to 0.8 microns wide and approximately 2.0 to 4.0 microns long. Its colony is smooth and moist, white round dot. No spore and flagellum. The strain YH-1 was identified as Lactobacillus hilgardii (Lactobacillus hilgardii) by 16S rDNA gene sequence and phylogenetic analysis. Lactobacillus hilgardii (Lactobacillus hilgardii) YH-1 was deposited at the China center for type culture Collection at 6/11/2020 at the deposition address: china Wuhan university, the preservation number is CCTCCNO: m2020203.
(3) Acid and bile salt resistance of YH-1
1) Adjusting the pH of the MRS culture medium to 1.5, 2.0, 2.5, 3.0, 3.5, 4.0 and 4.5 respectively by hydrochloric acid, respectively inoculating lactobacillus hilgardii YH-1 into the culture medium according to the inoculation amount of 5%, taking the MRS liquid culture medium without acid as a control, standing and culturing at 37 ℃ for 3h, respectively taking the bacteria liquid of the acid treatment group and the bacteria liquid of the control group for plate counting, and calculating the survival rate after acid treatment.
2) Adjusting the mass fractions of bile salts of an MRS culture medium to be 0.1%, 0.2%, 0.3% and 0.4% respectively by using pig bile salts, respectively inoculating lactobacillus hilgardii YH-1 into the culture media according to the inoculation amount of 5%, taking an MRS liquid culture medium without adding pig bile salts as a control, performing static culture at 37 ℃ for 3 hours, respectively taking bacteria liquid of an acid treatment group and a control group for plate counting, and calculating the survival rate after the acid treatment.
3) As a result: the following experimental results show that lactobacillus hilgardii YH-1 has better acid resistance and cholate resistance and has the potential of good probiotic performance. Lactobacillus hilgardii YH-1 pressed 106～107The CFU/g concentration can be added into different animal feeds to improve animal qualityThe feed additive has the advantages of improving intestinal health, improving the immunity of animals, improving the production performance of the animals and having good probiotic performance.
Acid and cholate resistance experiment result of lactobacillus hilgardii YH-1
Example 2:
method for preparing ferment by fermenting pineapple juice with Lactobacillus hilgardii (Lactobacillus hilgardii) YH-1:
(1) inoculating Lactobacillus hilgardii (Lactobacillus hilgardii) YH-1 preserved on the slant into a sterilized and cooled MRS liquid culture medium, and performing static culture at 37 ℃ for 24h to prepare Lactobacillus hilgardii YH-1 seed liquid for later use;
(2) cleaning 200 kg of fresh pineapples (without peeling) with sterile water, putting the pineapples into a juicer, adding 60 kg of sterile water into the pineapples, filtering the pineapple juice with gauze after juicing, and collecting the juice for later use;
(3) mixing 200 kg of molasses with 800 kg of water, adding 2 kg of calcium chloride, 3 kg of ferrous chloride, 8 kg of ammonium sulfate and 5 kg of dipotassium hydrogen phosphate into the mixture, uniformly mixing, and sterilizing at 121 ℃ for 20 min;
(4) adding 100 kg of prepared pineapple juice into the sterilized solution cooled to room temperature obtained in the step (3), and uniformly mixing to obtain a fermentation culture medium;
(5) adding 50 kg of the lactic acid bacteria seed liquid prepared in the step (1) into the fermentation medium prepared in the step (4), and standing and culturing for 36h at 37 ℃ (the total acid of the fermentation liquid is not increased any more, namely the fermentation end point);
(6) adding 0.5 kg of protease, 0.5 kg of cellulase, 0.5 kg of xylanase, 0.5 kg of mannanase, 0.5 kg of glucose oxidase, 1 kg of chitosan, 4 kg of soybean protein and 5 kg of calcium propionate into the culture medium after the fermentation is finished, and uniformly mixing to obtain the finished product.
The ferment prepared by the method comprises 10.2% of total acid and 2.0 × 10% of lactobacillus9CFU/mL, protease: 3000U/mL, cellulase: 1000U/mL, xylanase: 1000U/mL, mannanase: 1000U/mL; glucose oxidase: 1000U/mL.
Example 3: the difference between the acidity and the flavor components of the pineapple juice fermented by different lactic acid bacteria:
according to the method of the embodiment 3, the lactobacillus hilgardii YH-1 is replaced by lactobacillus plantarum CICC 20265, enterococcus faecalis CICC 21869, pediococcus acidilactici CICC 10146, lactobacillus casei CICC 20286, lactobacillus delbrueckii CICC 20247 and lactobacillus acidophilus CICC 6092, and the rest steps are the same, and the fermentation time is 36-48 h (taking the condition that the total acid of the fermentation liquid is not increased any more as the fermentation end point). The total acid of the bromelain prepared after fermenting the pineapple juice with different lactic acid bacteria after the fermentation was completed was measured, and the results are shown in the following table. The results in the following table show that the lactobacillus hilgardii not only has the fastest fermentation speed but also has the best acid production performance in the pineapple juice fermentation medium.
| Bacterial strain | Fermentation time (h) | Total acid |
| Lactobacillus hilgardii YH-1 | 36 | 10.2％ |
| Lactobacillus plantarum CICC 20265 | 48 | 5.2％ |
| Enterococcus faecalis CICC 21869 | 40 | 5.4％ |
| Pediococcus acidilactici CICC 10146 | 45 | 6.2％ |
| Lactobacillus casei CICC 20286 | 42 | 6.3％ |
| Lactobacillus delbrueckii CICC 20247 | 46 | 5.7％ |
| Lactobacillus acidophilus CICC 6092 | 43 | 8.2％ |
The flavor components of the bromelain prepared by fermenting different lactic acid bacteria in example 3 were identified by gas chromatography-mass spectrometry (GC-MS). The gas chromatography was an Agilent 6890A, the mass spectrum was 5975C, DB-5 column (30 m.times.0.25 mm. times.0.25 μm). The temperature rising procedure is as follows: maintaining the furnace temperature at 39 ℃ for 10min, then heating to 100 ℃ at 4 ℃/min, and maintaining for 3 min; then the temperature is raised to 180 ℃ at the rate of 3 ℃/min, and then the temperature is raised to 220 ℃ at the rate of 4 ℃/min and kept for 3 min. Mass transfer line temperature 250 ℃, ion source temperature 230 ℃, ion flux 70e V; the scanning range is 50-300 m/z. The results are shown in the following table. The results in the following table show that the bromelain prepared by lactobacillus hilgardii fermentation not only has more ester compounds, but also has more abundant content, so that the bromelain prepared by lactobacillus hilgardii fermentation has better flavor.
Bromelain products prepared by fermenting different lactic acid bacteria and having various flavor component contents
Example 4: the application of the ferment prepared by fermenting the lactobacillus hilgardii YH-1 in preparing the piglet feed additive comprises the following steps:
the bromelain prepared by fermenting pineapple juice with different lactic acid bacteria is applied to a certain pig farm in Hainan during piglet production, and the experiment is divided into a control group (pig feed without the added bromelain) and a bromelain group (the bromelain prepared by fermenting different lactic acid bacteria is fed with a mixed feed according to the proportion of 0.5%). After the piglets are transferred to a nursery house, a pre-feeding period of 3 days is carried out, and after 12 hours of fasting, the piglets are weighed and enter a formal test period. The test pigs adopt free drinking water and free ingestion, and the test is divided into a front stage and a later stage for 28 days. Water and food were freely drunk during the test period. During the test period, the feed intake of each repetition and the final mass of each test period were recorded, 5mL (3 pigs per repetition) of the blood was collected from the anterior vena cava after the completion of the test (i.e., day 29) by calculating the Average Daily Gain (ADG), the Average Daily Feed Intake (ADFI) and the feed mass ratio (F/G), and after standing at room temperature for 30min while inclining, the blood was centrifuged at 8000r/min for 10min, and the supernatant serum was taken and dispensed into 2mL centrifuge tubes and stored at-20 ℃ for testing. Serum superoxide dismutase (SOD), total antioxidant capacity (T-AOC) and IgG were measured. The influence of the bromelain prepared by fermentation of different lactic acid bacteria on the production performance, the antioxidant function and the immune function of piglets is shown in the following table. The results in the following table show that the bromelain prepared by lactobacillus hilgardii can improve the production performance of piglets, improve the serum IgG level, the oxidation resistance and the health level, and the growth promotion performance, the enhanced oxidation resistance and the immune function of the bromelain are obviously superior to those of the bromelain prepared by fermenting other lactic acid bacteria.
Influence of pineapple enzymes prepared by fermentation of different lactic acid bacteria on piglet production performance, antioxidant function and immune function
Example 5 Bromelain control of Vibrio paraguayensis in shrimp farming
The pineapple ferment prepared in the example 2 is added into prawn feed, and after being uniformly mixed, the prawn feed is used for feeding prawn, and a research experiment of controlling vibrio in the prawn culture process is carried out. Using Litopenaeus vannamei 108The test was repeated 3 times, with a total of 4 groups, namely, a control group (fed without enzymes), a1 group (fed with 0.1% enzymes), a2 group (fed with 0.3% enzymes), and A3 group (fed with 0.5% enzymes), with a water temperature of 25(± 2) ° c, 24 hours of aeration, with a total of 4 times per day (8:30, 12:30, 16:30, 20:30), with a total of 10L per tank, with a total of 10 shrimp, after soaking for half an hour, of CFU/mL vibrio alginolyticus, the water tank was filled with water, and the water quality was maintained, with a total of 3% enzymes per tank, with a total of 4 water temperatures per test, with a total of 25 ± 2 ℃ water temperature, with 24 hours of aeration, with a total of 4 times per day (8: 30: 12:30, 16:30, 20:30) of daily bait, with a total of 3% to 6% of shrimp weight, with a total of bait per day, with a total of 1d per tank, with a total of water sample taken before changing water, with a total of 1d, with a total of no more bacteria added, with a total of pineapple enzymes, and with a total of the total of three days.
Effect of Bromelain on Vibrio paraguatus in prawn culture Process
Example 6:
the enzyme provided by the invention has the following quality stability:
the pineapple ferment prepared in example 2 was stored at room temperature, and the main technical index of the product was measured every 60 days, and the results are shown in the following table. As can be seen from the results shown in the following table, the product was stable during preservation, and in particular, the enzyme activities of the various enzymes were maintained at 90% after 180 days at room temperature.
The change of technical index of the product in the preservation process
Claims (8)
1. Isolated Lactobacillus hilgardii (L.) useful as a probiotic in the intestinal tract of an animalLactobacillus hilgardii) The preservation number of the lactobacillus hilgardii is CCTCC No: m2020203.
2. Fruit ferment produced by fermentation with lactobacillus hilgardii according to claim 1.
3. Use of lactobacillus hilgardii according to claim 1 for the preparation of fruit ferments.
4. Use of the fruit ferment of claim 2 for the preparation of an animal feed additive.
5. The fruit ferment of claim 2, or the use of claim 2 or 3, wherein the fruit is one or a mixture of two or more of pineapple, mulberry and green orange.
6. The fruit ferment of claim 2, wherein the fermentation medium used in the fermentation process is:
the mass ratio of the molasses to the water is 0.01-0.5: 1, calcium chloride accounting for 0.01 to 5 percent of the total mass of the molasses and the water, ferrous chloride accounting for 0.01 to 5 percent of the total mass of the molasses and the water, ammonium sulfate accounting for 0.01 to 5 percent of the total mass of the molasses and the water, dipotassium hydrogen phosphate accounting for 0.01 to 5 percent of the total mass of the molasses and the water, fruit juice accounting for 5 to 25 percent of the total mass of the molasses and the water, and natural pH.
7. The application of claim 4, the application process comprising:
(1) mixing molasses and water according to the ratio of 0.01-0.5: 1, mixing, adding 0.01-5% of calcium chloride, 0.01-5% of ferrous chloride, 0.01-5% of ammonium sulfate and 0.01-5% of dipotassium hydrogen phosphate into the mixture, sterilizing and cooling to room temperature after uniformly mixing, and adding 5-25% of fruit juice of the total mass of molasses and water to obtain a fermentation culture medium;
(2) inoculating lactobacillus hilgardii according to the inoculation amount of 1-9%Lactobacillus hilgardii) YH-1, and standing and culturing the seed liquid at 35-38 ℃ for 24-40 h; the mass percentage of the inoculation amount in the step is calculated according to the total mass of the fermentation medium;
(3) adding 0.01-5% of protease, 0.01-5% of cellulase, 0.01-5% of xylanase, 0.01-5% of mannase, 0.01-5% of glucose oxidase, 0.01-5% of chitosan, 0.01-5% of soybean protein and 0.01-5% of calcium propionate into a culture medium after fermentation is finished, and uniformly mixing to obtain the microbial fertilizer; the mass percentage in the step is calculated according to the total mass of the culture medium after the fermentation is finished;
the fruit juice is one or a mixture of two or three of pineapple juice, mulberry juice or green orange juice.
8. The use of claim 7, wherein the fruit juice is prepared by washing fruit with sterile water, adding sterile water, squeezing, and filtering; the mass ratio of the fruits to the water is 1: 0.05 to 1.
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