WO2019161631A1 - 一种罗伊氏乳杆菌ss23-52及其干粉发酵剂的制备方法与在纯种益生菌酸奶中的应用 - Google Patents

一种罗伊氏乳杆菌ss23-52及其干粉发酵剂的制备方法与在纯种益生菌酸奶中的应用 Download PDF

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WO2019161631A1
WO2019161631A1 PCT/CN2018/094621 CN2018094621W WO2019161631A1 WO 2019161631 A1 WO2019161631 A1 WO 2019161631A1 CN 2018094621 W CN2018094621 W CN 2018094621W WO 2019161631 A1 WO2019161631 A1 WO 2019161631A1
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fermentation
lactobacillus reuteri
dry powder
cfu
yoghurt
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PCT/CN2018/094621
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English (en)
French (fr)
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郝红炜
刘慧�
张红星
谢远红
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富乐顿生物工程科技（北京）有限公司
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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
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
    • A23C9/00—Milk preparations; Milk powder or milk powder preparations
    • A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
    • A23C9/123—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt
    • A23C9/1234—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt characterised by using a Lactobacillus sp. other than Lactobacillus Bulgaricus, including Bificlobacterium sp.
    • 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
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
    • A23C2220/00—Biochemical treatment
    • A23C2220/20—Treatment with microorganisms
    • A23C2220/202—Genetic engineering of microorganisms used in dairy technology
    • 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
    • A23V2400/173—Reuteri
    • 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
    • C12R2001/225—Lactobacillus

Definitions

  • the invention relates to the preparation of a space of Lactobacillus reuteri SS23-52 and its dry powder starter and its application in pure probiotic yogurt.
  • Lactobacillus reuteri cells ranges from short rod-shaped, long rod-shaped to filamentous, single or short-chain, Gram-positive oxygen-resistant or microaerobic bacteria, cultured in MRS.
  • the size of the colony on the base plate is 1-2 mm, the surface is smooth and humid, the edge is irregular, flat, translucent, grayish white, and the lactic acid fermentation can be carried out to ferment glucose to produce lactic acid, acetic acid, ethanol and CO 2 .
  • the bacteria often inhabit the intestines of humans and animals, and can produce extracellular polysaccharides during growth and metabolism, so that they have strong adhesion to intestinal mucosa, antagonize colonization of intestinal pathogenic bacteria, and regulate intestinal bacteria.
  • the bacterium can use glycerol to produce a special bacteriostatic substance, reuterin, in the process of growth and metabolism. Its main component is 3-hydroxypropionaldehyde (3-HPA), which is non-protein wide.
  • Antibacterial substances can inhibit the growth of gastrointestinal pathogens such as Escherichia, Shigella, Salmonella, Listeria, Vibrio, Clostridium, Staphylococcus, and Helicobacter pylori, and avoid intestinal diseases. It is effective in preventing and treating diarrhea in children.
  • Space microbes are subject to mutagenic effects such as space microgravity effects, high vacuum, extreme temperature differences, weak magnetic fields, and high-energy particles (electrons, protons, heavy ions), which can significantly increase the frequency of mutations and cause gene mutations, which will lead to their biological traits. (such as individual morphology, colony characteristics, physiological and biochemical characteristics, immunogenicity, etc.), fermentation production performance (such as biomass, product amount, enzyme activity, potency, fermentation rate, etc.) changed.
  • mutagenic effects such as space microgravity effects, high vacuum, extreme temperature differences, weak magnetic fields, and high-energy particles (electrons, protons, heavy ions), which can significantly increase the frequency of mutations and cause gene mutations, which will lead to their biological traits. (such as individual morphology, colony characteristics, physiological and biochemical characteristics, immunogenicity, etc.), fermentation production performance (such as biomass, product amount, enzyme activity, potency, fermentation rate, etc.) changed.
  • Lactobacillus reuteri to the compound probiotic yoghurt based on the subspecies is obviously not as good as the pure L. reuteri yoghurt. Therefore, the present invention for the selection of the space L. reuteri and its application in pure probiotic yoghurt fills the research gap of the space food microbial engineering bacteria. Therefore, the fermentative production methods for the space Lactobacillus reuteri and its pure probiotic yoghurt have not been reported at home and abroad.
  • the present invention firstly provides a strain which can be used for preparing pure probiotic yoghurt - Lactobacillus reuteri Fullarton-H-SS23-52, which may be referred to as Lactobacillus reuteri SS23-52 for short.
  • the deposit number of the General Microbiology Center of the China Microbial Culture Collection Management Committee is CGMCC No. 15152.
  • the present invention also provides a microbial agent whose active ingredient is Lactobacillus reuteri Fullarton-H-SS23-52.
  • the microbial agent is a culture obtained by culturing Lactobacillus reuteri Fullarton-H-SS23-52.
  • the microbial agent may specifically be a culture obtained by inoculating Lactobacillus reuteri Fullarton-H-SS23-52 in MRS liquid medium.
  • the live bacteria of the L. reuteri may specifically be 7.5 ⁇ 10 9 CFU/mL.
  • the microbial agent can also include a carrier.
  • the carrier can be a solid carrier or a liquid carrier.
  • the solid carrier may be a sugar alcohol, a protein or a vitamin; the sugar alcohol carrier may be at least one of sea bath sugar, lactose, sucrose, maltodextrin, maltose, sucrose, fructose, mannitol and sorbitol.
  • One of the protein carriers is at least one of skim milk powder, whey powder, yeast powder and casein; the vitamin carrier may be vitamin C and/or vitamin E.
  • the liquid carrier can be glycerin, vegetable oil or water.
  • the active ingredient may be present in the form of cultured living cells, fermentation broth of living cells, filtrate of cell culture, or a mixture of cells and filtrate.
  • the dosage form of the composition can be in a variety of dosage forms such as liquids, emulsions, suspensions, powders, granules, wettable powders or water-dispersible granules.
  • the present invention also protects a dry powder starter obtained by mixing Lactobacillus reuteri Fullarton-H-SS23-52 or a fermentation product thereof with a lyoprotectant and lyophilizing.
  • the fermentation product may specifically be a living cell after fermentation.
  • the number of viable bacteria of the Lactobacillus reuteri Fullarton-H-SS23-52 is as follows (a1) or (a2) per 1 g of the dry powder starter:
  • the lyoprotectant contains the following components (b1) or (b2):
  • the lyoprotectant consists of 5 g/100 mL to 20 g/100 mL maltodextrin, 5 g/100 mL to 20 g/100 mL skim milk powder and a solvent; the solvent is water, more specifically distilled water.
  • the lyoprotectant consists of 5 g / 100 mL maltodextrin, 10 g / 100 mL skim milk powder and a solvent; the solvent is water, more specifically distilled water.
  • the lyoprotectant is autoclaved (specifically, it can be achieved by autoclaving for 15 min at 0.07 MPa).
  • the fermentation product of Lactobacillus reuteri Fullarton-H-SS23-52 is a fermentation obtained by fermenting Lactobacillus reuteri Fullarton-H-SS23-52 in liquid MRS medium. product.
  • the fermentation product may specifically be a living cell after fermentation.
  • the content of the Lactobacillus reuteri Fullarton-H-SS23-52 is as follows (d1) or (d1) or (d1):
  • the Lactobacillus reuteri Fullarton-H-SS23-52 may be present in any of the above-mentioned microbial agents, and the introduction of the bacterial agent into the liquid MRS medium is used to introduce L. reuteri ( Lactobacillus reuteri) The purpose of Fullarton-H-SS23-52.
  • the bacteria The inoculum of the agent can be (d4) or (d5) or (d6):
  • the temperature of the fermentation is (e1) or (e2) or (e3):
  • the fermentation time is (f1) or (f2) or (f3):
  • the pH of the fermentation system was maintained at 6.8 (specifically by adding 20 g/100 mL of NaOH solution).
  • the stirring speed of the fermentation may specifically be 120 to 150 r/min.
  • the fermentation product of any of the above may specifically be a precipitate (bacteria) obtained by centrifuging the fermented fermentation system.
  • the centrifugation conditions can be specifically centrifuged at 4 ° C, 4000 r / min for 20 min.
  • the mixing ratio of the fermented living cell body precipitation product and the lyoprotectant can be specifically: the living cell body precipitate product obtained after centrifugation per 100 mL of the fermentation system is uniformly mixed by adding 10 mL of the lyoprotectant.
  • the lyophilization may specifically pre-freeze the mixture of the fermentation product and the lyoprotectant at -80 ° C for 2 to 4 hours to a completely frozen state, and then freeze-dry for 36 to 48 hours at -55 ° C and a vacuum of 0.13 mBar. Completely dry.
  • the use of the dry powder starter can be the preparation of yogurt.
  • the present invention also protects Lactobacillus reuteri Fullarton-H-SS23-52, or any of the above-described bacterial agents, or the use of any of the above dry powder starters in the preparation of yoghurt.
  • the present invention also protects a method for preparing a yogurt, comprising the steps of: adding Lactobacillus reuteri Fullarton-H-SS23-52 to the raw milk, or any of the above-mentioned bacterial agents, or above Any of the dry powder starters is fermented to obtain yoghurt.
  • the content of the Lactobacillus reuteri Fullarton-H-SS23-52 is as follows (c1) or (c2) or (c3):
  • Lactobacillus reuteri Fullarton-H-SS23-52 to the raw milk can be achieved by adding any of the above dry powder starters to the raw milk.
  • the The inoculum of the dry powder starter can be (c4) or (c5) or (c6):
  • the fermentation system further includes cotton white sugar or sucrose.
  • the preparation method of the yoghurt may specifically be: heating raw material milk (specifically, Tetra Pillow packaging, net content: 240 mL/bag, Inner Mongolia Yili Industrial Group Co., Ltd.) to 60 ° C, and adding 5.5 g/100 mL of white sugar (sucrose) The mass content is 95%), continue to heat to 90 ° C for 5 to 10 minutes, and cooled to 37 ° C to obtain a sterilized raw milk.
  • the inoculum of 1.0 ⁇ (mass by volume) will be used to treat any of the above Roy. Lactobacillus reuteri Fullarton-H-SS23-52 dry powder starter was added, stirred evenly, and fermented at 37 ° C until solidification to obtain yoghurt.
  • the yoghurt containing the Lactobacillus reuteri Fullarton-H-SS23-52 has a live bacterial count of 3.2 ⁇ 10 9 CFU/mL, an acidity of 54.71°T, and a curd time of 3.5 h.
  • the curd time of the commercial yoghurt was produced by the symbiotic action of Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus (fermentation time was 5-6 h).
  • any of the above raw milks is mammalian milk or a liquid dairy product obtained from the mammalian milk, which can be used to prepare yoghurt.
  • the raw milk may specifically be cow's milk.
  • the invention also protects the yogurt prepared by any of the methods described above.
  • Raw milk The raw milk is pure milk (the total bacterial count is generally less than 10 4 CFU/mL, no antibiotics and disinfectants, and it is not suitable to use mastitis).
  • the raw milk is centrifuged to remove white blood cells and other visible impurities in the milk.
  • the main component indicators of raw milk should meet the national food safety standard GB 5408-85.
  • the total dry matter should be no less than 11.5%, and the fat content is roughly adjusted to four according to the product: 3.2%, 2.5%, 1.0% and ⁇ 0.1%, by removing the cream or adding 1% to 3% of skimmed milk powder. Or cream to adjust the total dry matter or fat content.
  • the preheated raw milk is homogenized in a homogenizer under a pressure of 8-10 MPa.
  • the purpose is to make the milk coagulate evenly, the texture is more delicate and smooth, and the fat globule can be made smaller to prevent the fat from floating.
  • Cooling The sterilized raw milk is rapidly cooled to 37 ° C until inoculation.
  • Inoculation Inoculate the raw milk with the dry powder fermentation agent of Lactobacillus reuteri SS23-52 (the number of viable bacteria of Lactobacillus reuteri SS23-52 is 4.4 ⁇ 10 10 CFU/g), and the inoculation amount is 1.0. ⁇ (mass volume and concentration, that is, the number of viable cells of Lactobacillus reuteri SS23-52 in the fermentation system is 4.4 ⁇ 10 7 CFU/mL).
  • Insulation fermentation The small plastic container is placed in the fermentation chamber to maintain the fermentation temperature at 37 °C. When the acidity of the fermented milk reaches 55 to 70 °T and the milk coagulation property is good, the fermentation is mature. The fermentation time is 3 to 4 hours.
  • Cooling The container containing the yogurt is taken out of the fermentation chamber and rapidly cooled to below 10 °C with cold air.
  • Refrigeration and post-cooking The chilled yogurt should be stored in a 0 ⁇ 5 ° C freezer until it is consumed.
  • any of the above yoghurts may specifically be probiotic yoghurt, more specifically, Lactobacillus reuteri pure probiotic yoghurt.
  • the invention utilizes the Tiangong 2 and the Shenzhou 11 spacecraft to carry the Lactobacillus reuteri after returning to the ground and is subjected to space mutagenesis, and uses the ground original Lactobacillus reuteri as a control to select and ferment the positive mutation.
  • the strain space of Lactobacillus reuteri SS23-52 by optimizing high-density fermentation conditions and lyoprotectant, developed a dry powder starter prepared by the strain, which can be used to develop and produce pure probiotic yoghurt.
  • the pure probiotic yoghurt obtained by using the space of Lactobacillus reuteri SS23-52 of the present invention has rich flavor of fried wheat, has fine taste and lubrication, moderate sweet and sour taste, acidity of 54.71 °T, curd firm, whey Less precipitation, shortening the curd time to 3.5h, breaking the curd time of producing common commercial yoghurt by using the symbiotic action of Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus (fermentation time is 5-6h) .
  • Biomaterials preservation unit name China Microbial Culture Collection Management Committee General Microbiology Center
  • Figure 1 shows the process of producing pure probiotic yoghurt from Lactobacillus reuteri SS23-52.
  • Lactobacillus reuteri GS23 American type culture collection, number: ATCC23272.
  • the MRS liquid medium in the following examples consists of a solute and a solvent.
  • the solvent is distilled water, and the solute and its concentration are: casein ⁇ 10 g / L, beef extract 10 g / L, yeast powder 5 g / L, glucose 10 g / L, Sodium acetate 5g/L, diammonium citrate 2g/L, Tween-80 1mL/L, K 2 HPO 4 2g/L, MgSO 4 ⁇ 7H 2 O 0.2g/L, MnSO 4 ⁇ H 2 O 0.05g/ L, pH 6.8.
  • the MRS solid medium was a medium obtained by adding 1.7 g/L of agar powder to MRS liquid medium.
  • Example 1 Screening of a spatial strain of Lactobacillus reuteri
  • Lactobacillus reuteri GS23 was transported back by Tiangong 2 and Shenzhou 11 spacecraft to obtain space-mutant strains. 115 strains were isolated and purified, and the strains were labeled SS23-1 to SS23-. 115.
  • Lactobacillus reuteri GS23 and strain SS23-1 to SS23-115 glycerol storage tubes frozen in a -80 ° C freezer were placed in 5 mL MRS liquid medium at 2% to 3%, respectively, and cultured at 37 ° C. 16h, after three consecutive generations of activation, used for subsequent experiments.
  • the liquid MRS culture of Lactobacillus reuteri GS23 and strains SS23-1 to SS23-115 obtained in the second step was inserted into a 5 mL skim milk tube at a 2% inoculation amount, and cultured at 37 ° C until After the milk was coagulated, the curd time of each strain (F1 generation) was recorded, and the condition of the curd was described. Then continue to operate for 3 generations (F2-F4), record the curd time and curd of each generation (as shown in Table 1), select the curd time is shorter (the fermented milk is faster), and the curd is firm. The whey precipitates less fermented strains.
  • Lactobacillus reuteri GS23 has a longer curd time in the skim milk test tube, and it usually takes 2 to 3 days to coagulate the milk. Only the curd time of SS23-12, SS23-22, SS23-24, SS23-27, SS23-30, SS23-36, SS23-38, SS23-46, SS23-52, SS23-84, SS23-88 strains was obvious Shortened, in which the curd time of SS23-12, SS23-27, SS23-36, SS23-38, SS23-46, SS23-52, SS23-84, SS23-88 space L. reuteri was shortened to 7h, and condensation The milk is firm, indicating that the eight strains of L. reuteri have positive mutations in related genes under space environment conditions.
  • yogurt yoghurt fermentation conditions: 3% inoculum inoculum, 5g/100mL in cotton sugar, 37°C
  • the results of the determination of the viability of the fermenting agent of Lactobacillus reuteri and the sensory evaluation results of the yogurt were shown in Tables 4 and 5.
  • the curd time of the fermenting agent using SS23-36, SS23-84 and SS23-88 strains is 11-12 h, and the yogurt curd time is 7 h; while SS23-12, SS23-27, SS23-38
  • the curdifying time of SS23-46 and SS23-52 strains was 7h, the curd time of yoghurt was 3 ⁇ 4h, and the acidity and viable count were higher.
  • the SS23-27 strain has the highest sensory score for making yogurt, followed by SS23-88, SS23-52, SS23-46, SS23-12, SS23-38 strains, with rich fried wheat flavor and moderate sweet and sour taste.
  • the palate is fine and smooth, the curd is firm, and the whey is less precipitated. Therefore, the strains SS23-27, SS23-88, SS23-52, SS23-46, SS23-12, and SS23-38 are strains excellent in fermented milk.
  • the SS23-12, SS23-27, SS23-36, SS23-38, SS23-46, SS23-52, SS23-84, SS23-88 strains with excellent fermentation performance obtained by the above re-screening were continuously dried in 5 mL of liquid MRS medium. Passage for 50 generations (inoculation amount 2% to 3%, culture at 37 ° C for 16h), and then into the 5mL skim milk tube in 2% of the inoculum, culture at 37 ° C until the milk solidifies, record the curd time and curd state . As shown in Table 6, the continuous operation of the 6th generation in the milk, the 50-generation passage of the L. reuteri curd time and the curd state results are shown in Table 6.
  • SS23-52 strain is the best strain with the best genetic stability and the best performance of fermented milk, which can be the most potential strain for developing functional fermented dairy products.
  • the SS23-52 strain was subjected to Gram staining, and the results showed that the SS23-52 strain was a Gram-positive bacterium. Morphological observation, the strain of SS23-52 strain is short rod-shaped, long rod-shaped to filamentous, ranging from solitary or short-chain. On the MRS medium plate, the colony size is 1-2 mm and the surface is smooth. Wet, irregular edges, flat, translucent, grayish white.
  • the 16S rDNA of the SS23-52 strain was detected, and the sequencing result is shown in Sequence 1 of the Sequence Listing.
  • the 16s rDNA identification showed that the similarity between SS23-52 strain and Lactobacillus reuteri reached 100%. After morphological and 16S rDNA identification, it was confirmed that the SS23-52 strain belongs to Lactobacillus reuteri.
  • the SS23-52 strain was named Lactobacillus reuteri Fullarton-H-SS23-52 and deposited on January 2, 2018 in the General Microbiology Center of China Microbial Culture Collection Management Committee (CGMCC; Address: Beijing No. 3, Beichen West Road, Chaoyang District, No. 3, Institute of Microbiology, Chinese Academy of Sciences; Zip Code: 100101), the deposit number is CGMCC NO.15152.
  • Lactobacillus reuteri Fullarton-SS23-52 is abbreviated as Lactobacillus reuteri SS23-52.
  • the Lactobacillus reuteri SS23-52 glycerol preservation tube was inserted into 5 mL MRS liquid medium at 2% to 3% (volume percent concentration), and activated at 37 ° C for 16 h.
  • the activated MRS tube culture was activated.
  • the inoculation amount of 2% (volume percent concentration) was transferred into a triangular flask containing 200 mL of sterilized MRS medium, and cultured at 37 ° C for 16 h to obtain a space fermentation agent of Lactobacillus reuteri SS23-52, and the fermentation agent was tested.
  • the number of live bacteria of L. eutropha was 7.5 ⁇ 10 9 CFU/mL.
  • the method for detecting the space of Lactobacillus reuteri SS23-52 starter taking 1 mL of the starter into 99 mL of sterilized physiological saline, and treating with a tapping homogenizer at a speed of 8000 to 10000 r/min for 2 minutes, fully After shaking, a uniform dilution of 10 -2 was made. Dilute 10 mL in 10 mL sterilized saline to 10 -8 , take 1 mL of 10 -6 to 10 -8 dilutions in a sterile dish, and pour into MRS solid medium dissolved and cooled to 46 °C.
  • the plate was quickly swirled gently, and the medium was thoroughly mixed with the bacterial solution, and each dilution was repeated 3 times.
  • the MRS solid medium was injected into a sterilized dish supplemented with 1 mL of sterile physiological saline as a blank control. After the medium is solidified, the plate is inverted, and cultured in a (36 ⁇ 1) °C incubator (48 ⁇ 2) h, and counted after the colony grows.
  • the space L. reuteri SS23-52 starter prepared in the first step was inoculated in 200 mL of MRS liquid medium according to a 3% (volume percent concentration) inoculum, the initial pH of the fermentation system was 6.8, and the fermentation system was at different fermentation temperatures ( Fermentation at 31 ° C, 34 ° C, 37 ° C, 40 ° C, 43 ° C) for 16 h, the concentration of live bacteria in the fermentation broth was tested. The number of viable bacteria in the fermentation broth was measured by pouring plate culture using MRS agar medium, and each dilution was repeated three times. Table 7 shows the results of viable cells in the fermentation broth of different fermentation temperatures of Lactobacillus reuteri SS23-52.
  • Temperature is an important factor affecting the biomass production in high-density fermentation broth. As can be seen from Table 7, when the fermentation temperature is between 31 and 37 ° C, the number of live bacteria in the fermentation broth gradually increases, and when the fermentation temperature is between 37 and 43 ° C, the number of live bacteria in the fermentation broth gradually decreases.
  • the suitable fermentation temperature of Lactobacillus reuteri SS23-52 is between 34 and 40 °C.
  • the temperature of 37 °C is the highest in the fermentation broth, which is 8.57 ⁇ 10 9 CFU/mL. Therefore, the fermentation temperature of the space L. reuteri SS23-52 was determined to be 37 °C.
  • the space-prepared L. reuteri SS23-52 starter prepared in step 1 was inoculated into 200 mL of MRS liquid medium according to different inoculum amounts (1%, 2%, 3%, 4%, 5%, volume percent concentration), and fermented.
  • the initial pH of the system was 6.8, and the fermentation system was fermented at 37 ° C for 16 h to detect the concentration of viable bacteria in the fermentation broth.
  • the number of viable bacteria in the fermentation broth was measured by pouring plate culture using MRS agar medium, and each dilution was repeated three times.
  • Table 8 shows the results of viable cells in the fermentation broth of different doses of L. reuteri SS23-52.
  • the amount of inoculum is determined by the rate of propagation and biomass production of the production strain in the fermentor. If the inoculation amount is too low, the bacterial growth rate is slow; if the inoculum is too high, the nutrients in the culture medium are too fast, resulting in a decrease in the biological yield in the later stage. As can be seen from Table 8, when the inoculum amount is between 1% and 2%, the viable cell viable count is gradually increased, and when the fermentation temperature is between 2% and 5%, the viable liquid viable cell count is gradually decreased.
  • the suitable inoculum size of Lactobacillus reuteri SS23-52 is between 2% and 3%.
  • the viable liquid has the highest viable count, which is 7.72 ⁇ 10 9 CFU/mL. Therefore, it is determined that the fermentation inoculation amount of Lactobacillus reuteri SS23-52 is 2% (volume percent concentration, the viable cell number of the fermentation medium in the space of Lactobacillus reuteri SS23-52 is 7.5 ⁇ 10 9 CFU/mL, ie fermentation The viable cell count of the space L. reuteri SS23-52 in the system was 1.5 ⁇ 10 8 CFU/mL).
  • the space of Lactobacillus reuteri SS23-52 fermenting agent prepared in the first step was inoculated in 200 mL of MRS liquid medium according to the inoculation amount of 2% (volume percent concentration), the initial pH of the fermentation system was 6.8, and the fermentation system was fermented at 37 ° C.
  • the concentration of live bacteria in the fermentation broth was detected at different times (8h, 12h, 16h, 20h, 24h).
  • the number of viable bacteria in the fermentation broth was measured by pouring plate culture using MRS agar medium, and each dilution was repeated three times. Table 9 shows the results of the viable counts in the fermentation broth of the different fermentation time of Lactobacillus reuteri SS23-52.
  • the length of the fermentation time directly affects the biological yield of the bacteria in the fermentation broth. If the fermentation time is too short, the bacteria have not reached the logarithmic growth period where the vitality is extremely vigorous, and the bacterial biomass at the end of the logarithmic growth phase reaches the highest peak, so the shorter fermentation time will cause the biological yield to decrease; for example, the fermentation time Too long, the growth of the bacteria enters the decline phase, and the autolysis of the bacteria causes the biomass to decrease. It can be seen from Table 9 that when the fermentation time is between 8 and 16 hours, the number of live bacteria in the fermentation liquid gradually increases, and when the fermentation temperature is between 20 and 24 hours, the number of live bacteria in the fermentation liquid gradually decreases.
  • the suitable fermentation time of Lactobacillus reuteri SS23-52 was between 16 and 20 h.
  • the viable liquid had the highest viable count, which was 7.53 ⁇ 10 9 CFU/mL. Therefore, the fermentation time of the space Lactobacillus reuteri SS23-52 was determined to be 20h.
  • the high-density fermentation conditions of Lactobacillus reuteri SS23-52 were determined by single factor multi-level test: fermentation temperature was 37 ° C, inoculum volume was 2% (volume percent concentration, space in the starter)
  • the viable cell count of Lactobacillus johnsonii SS23-52 is 7.5 ⁇ 10 9 CFU/mL, that is, the number of viable cells of Lactobacillus reuteri SS23-52 in the fermentation system is 1.5 ⁇ 10 8 CFU/mL, and the fermentation time is 20h.
  • step two design three factors of fermentation temperature, starter inoculum and fermentation time [L 9 (3 4 )] orthogonal test (see Table 10), in 2L automatic fermenter, with 2L liquid MRS is the fermentation medium, and the fermentation system is controlled to pH 6.8 during batch fermentation (specifically by adding 20g/100mL NaOH solution to control the pH), the stirring speed is 120-150r/min, and the MRS agar medium is used to pour the plate.
  • the culture method was used to detect the number of viable bacteria in the fermentation broth, and each dilution was set to 3 repetitions.
  • the high-density fermentation process conditions were determined by the range analysis and K-value analysis of the test results.
  • the results of orthogonal experiment optimization of strain SS23-52 high-density fermentation conditions are shown in Table 11.
  • this experiment studies the high-density fermentation conditions of Lactobacillus reuteri, mainly from the indicator of the viable count of the fermentation broth.
  • the combination of high-density fermentation conditions of Lactobacillus reuteri SS23-52 was determined to be A2B2C2, ie the fermentation temperature was 37 ° C, and the inoculant dosage was 3% (volume percent concentration, in the starter
  • the viable cell count of Lactobacillus reuteri SS23-52 was 7.5 ⁇ 10 9 CFU/mL, ie the number of viable cells of Lactobacillus reuteri SS23-52 in the fermentation system was 2.3 ⁇ 10 8 CFU/mL), fermentation
  • the time is 16h, and the fermentation system is controlled at pH 6.8, and the stirring speed is 120-150r/min.
  • the space fermentation bacterium of Lactobacillus reuteri SS23-52 was transferred into 2L liquid MRS medium by 3% inoculum, stirred and fermented at 37 ° C for 16 h, and other fermentation conditions were compared.
  • the orthogonal test was the same, and the control conditions (fermentation temperature 37 ° C, inoculum amount 2%, fermentation time 20 h) were used as a control group, and the number of viable bacteria in the fermentation broth was measured. The results are shown in Table 12.
  • the space of Lactobacillus reuteri SS23-52 fermenting agent prepared in the first step of Example 2 is 3% (volume percent concentration, the number of viable bacteria in the space of the fermentation agent in the space of Lactobacillus reuteri SS23-52 is 7.5 ⁇ 10 9 CFU/mL, ie the number of viable cells of Lactobacillus reuteri SS23-52 in the fermentation system was 2.3 ⁇ 10 8 CFU/mL.
  • the inoculum was inoculated into a 5L automatic fermenter containing 2.4L liquid MRS medium.
  • the fermentation temperature was controlled at 37 ° C, the fermentation system pH 6.8, the stirring speed was 120-150 r/min, and the fermentation time was 16 h.
  • the fermentation broth after the fermentation in step 1 is divided into 8 parts, centrifuged at 4°C and 4000r/min for 20min, the supernatant is discarded, and the bacterial sludge is collected and uniformly mixed with the above 8 kinds of 30mL sterile lyoprotectants.
  • the bacterial sludge obtained after centrifugation of each 100 mL fermentation system can be uniformly mixed by adding 10 mL of lyophilized protective agent) to obtain a concentrated live bacterial preparation.
  • the number of viable bacteria in the concentrated live bacterial preparation was measured by pouring plate culture method using MRS medium, and each dilution was set to 3 times. The results are shown in Table 13 to determine the preferred combination of the lyoprotectant.
  • Method for detecting the number of viable bacteria in the concentrated live bacteria preparation before lyophilization Weigh 1 mL of the concentrated live bacterial preparation into 99 mL of sterilized physiological saline, and treat it with a tapping homogenizer at a speed of 8000 to 10000 r/min for 3 minutes. After sufficient shaking, make a uniform dilution of 10 -2 ; then dilute to 10 -9 in 10 times with 9 mL of sterile physiological saline, and take 1 mL of each dilution of 10 -7 to 10 -9 in a sterile dish.
  • MRS agar medium pour into about 15 mL of MRS agar medium which melted and cooled to 46 ° C, and gently swirl the plate to mix the medium and the bacterial solution thoroughly, repeating each dilution 3 times.
  • MRS agar medium was injected into a sterilized dish supplemented with 1 mL of sterile physiological saline as a blank control. After the medium is solidified, the plate is inverted, and cultured in a (36 ⁇ 1) °C incubator (48 ⁇ 2) h, and counted after the colony grows.
  • the concentrated live bacterial preparation prepared in the first step was pre-frozen at -80 ° C for 2 to 4 hours to a completely frozen state to obtain a pre-freezing live bacterial preparation.
  • the pre-frozen live bacterial preparation was lyophilized by a 6 L LABCONCO vacuum freeze dryer (USA) at -55 ° C under a vacuum of 0.13 mBar for 36 to 48 hours to a completely dry state to obtain a freeze-dried live bacterial preparation.
  • the number of live bacteria in the lyophilized preparation was measured by pouring plate culture method using MRS medium, and the survival rate of the strain was calculated, and each dilution was repeated 3 times. The results are shown in Table 13.
  • Method for detecting the number of viable bacteria in the live bacterial preparation after lyophilization Weigh 1 g of the freeze-dried live bacterial preparation, and add a quantitative sterilized physiological saline (the lyophilized live bacterial preparation is obtained by freeze-drying according to 30 mL of the concentrated live bacterial preparation, Calculate the amount of sterile physiological saline added to prepare a reduced-concentration live bacterial preparation), and shake it for 1 minute with a vortex shaker to obtain a uniform concentrated live bacterial preparation; take 1 mL of the concentrated live bacterial preparation and put it into 99 mL of sterile physiological saline.
  • MRS agar medium was injected into a sterilized dish supplemented with 1 mL of sterile physiological saline as a blank control. After the medium is solidified, the plate is inverted, and cultured in a (36 ⁇ 1) °C incubator (48 ⁇ 2) h, and counted after the colony grows.
  • the survival rate of the L. reuteri SS23-52 with the combination of the lyophilized protective agent No. 4 is the highest, which is 98.67%; the survival rate of the combination of the lyophilized protective agent No. 8 is the second, which is 97.67%;
  • the survival rate of the combination of No.1, No.2, No.3 and No.5 lyophilized protective agent was between 91.49 and 95.86, and the survival rate of the combination of No.6 and No.7 lyophilized protective agent was the smallest, which was 86.33% and 86.90%, respectively.
  • the lyophilized protective agent of Lactobacillus reuteri SS23-52 is better: 5g/100mL maltodextrin +10g/100mL skim milk powder, the survival rate of the strain can reach over 98%, freeze-dried starter The viable count was 4.4 ⁇ 10 10 CFU/g.
  • the optimal combination formula of the single-factor multi-level test to optimize the space of Lactobacillus reuteri SS23-52 lyoprotectant is: 5g/100mL maltodextrin +10g/100mL skim milk powder, the survival rate of the strain Up to 98% or more, the live bacteria of the freeze-dried starter is 4.4 ⁇ 10 10 CFU/g.
  • the space of Lactobacillus reuteri SS23-52 fermenting agent prepared in the first step of Example 2 is 3% (volume percent concentration, the number of viable bacteria in the space of the fermentation agent in the space of Lactobacillus reuteri SS23-52 is 7.5 ⁇ 10 9 CFU/mL, that is, the number of viable cells of Lactobacillus reuteri SS23-52 in the fermentation system was 2.3 ⁇ 10 8 CFU/mL.
  • the inoculum was inoculated into a 5L automatic fermenter containing 2L of liquid MRS medium.
  • the fermentation temperature was controlled at 37 ° C, the fermentation system pH 6.8, the stirring speed was 120-150 r/min, and the fermentation time was 16 h.
  • step 2 the fermentation broth was centrifuged at 4 ° C, 4000 r / min for 20 min, the supernatant was discarded, and the slime was collected.
  • the lyoprotectant is an aqueous solution containing 5g/100mL maltodextrin and 10g/100mL skim milk powder, and sterilized by autoclaving at 0.07Mpa for 15min.
  • the bacterial sludge prepared in the step 2 is uniformly mixed with the lyophilized protective agent prepared in the step 3, and pre-frozen at -80 ° C for 2 to 4 hours to a completely frozen state to obtain a pre-freezing live bacterial preparation.
  • the pre-frozen live bacterial preparation was lyophilized at -55 ° C under a vacuum of 0.13 mBar for 36-48 h to complete dryness to obtain a dry powder of Lactobacillus reuteri SS23-52.
  • Fermentation agent (the number of viable cells of Lactobacillus reuteri SS23-52 in the space dry yeast of R. reuteri SS23-52 was 4.4 ⁇ 10 10 CFU/g).
  • the sensory evaluation score of yoghurt prepared by dry powder starter with inoculum of 1.0 ⁇ was the highest, being 58.58 points, followed by 1.5 ⁇ and 2.0 ⁇ .
  • the inoculation amount and the sensory evaluation score were 58.58 and 57.55, respectively. Therefore, it is determined that the optimum dosage of the pure powder probiotic yoghurt addition space L. reuteri SS23-52 dry powder starter is 1.0 ⁇ .
  • fermentation temperature is 37 ° C
  • dry powder starter dosage is 1.0 ⁇ (mass volume thousand
  • concentration of live bacteria in the concentration of the spatial Lactobacillus reuteri SS23-52 dry powder starter in the Lactobacillus reuteri SS23-52 is 4.4 ⁇ 10 10 CFU/g, ie the space of Lactobacillus reuteri SS23-52 in the fermentation system.
  • viable count was 4.4 ⁇ 10 7 CFU/mL) and the curd time was 3.5 h.
  • the finished yoghurt has rich flavor of fried wheat, fine and lubricious taste, moderate sweet and sour taste, firm curd and less whey precipitation.
  • the number of live bacteria is 3.2 ⁇ 10 9 CFU/mL and the acidity is 54.71°T. Breaking through the symbiotic action of Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus to produce commercial yoghurt time (fermentation time is 5-6h).
  • Raw milk The raw milk is pure milk (the total bacterial count is generally less than 10 4 CFU/mL, no antibiotics and disinfectants, and it is not suitable to use mastitis).
  • the raw milk is centrifuged to remove white blood cells and other visible impurities in the milk.
  • the main component indicators of raw milk should meet the national food safety standard GB 5408-85.
  • the total dry matter should be no less than 11.5%, and the fat content is roughly adjusted to four according to the product: 3.2%, 2.5%, 1.0% and ⁇ 0.1%, by removing the cream or adding 1% to 3% of skimmed milk powder. Or cream to adjust the total dry matter or fat content.
  • the preheated raw milk is homogenized in a homogenizer under a pressure of 8-10 MPa.
  • the purpose is to make the milk coagulate evenly, the texture is more delicate and smooth, and the fat globule can be made smaller to prevent the fat from floating.
  • Cooling The sterilized raw milk is rapidly cooled to 37 ° C until inoculation.
  • Inoculation Inoculate the raw milk with the dry powder fermentation agent of Lactobacillus reuteri SS23-52 (the number of viable bacteria of Lactobacillus reuteri SS23-52 is 4.4 ⁇ 10 10 CFU/g), and the inoculum is 1.0 ⁇ .
  • the mass-volume concentration, the number of viable bacteria in Lactobacillus reuteri SS23-52 in the dry powder fermentation agent of Lactobacillus reuteri SS23-52 is 4.4 ⁇ 10 10 CFU/g, ie the space Roy's milk in the fermentation system
  • the viable cell count of Bacillus sp. SS23-52 was 4.4 ⁇ 10 7 CFU/mL).
  • Insulation fermentation The small plastic container is placed in the fermentation chamber to maintain the fermentation temperature at 37 °C. When the acidity of the fermented milk reaches 55 to 70 °T and the milk coagulation property is good, the fermentation is mature. The fermentation time is 3 to 4 hours.
  • Cooling The container containing the yogurt is taken out of the fermentation chamber and rapidly cooled to below 10 °C with cold air.
  • Refrigeration and post-cooking The chilled yogurt should be stored in a 0 ⁇ 5 ° C freezer until it is consumed.
  • the purpose is to prevent the yogurt from continuing to ferment and produce acid, which causes the pH to be too low, which affects the taste and prevents the contamination of the bacteria.
  • the post-ripening under cold storage conditions is conducive to the formation of yoghurt flavoring substances, and finally has a rich fried wheat flavor, delicate and lubricious taste, moderate sweet and sour taste, firm curd, and less pure yoghurt probiotic yoghurt.
  • the invention relates to the selection of the space Lactobacillus reuteri and its application in the pure probiotic yoghurt, and fills the research blank of the space food microbial engineering bacteria.
  • the raw material of the pure probiotic yoghurt of the invention is convenient, the fermentation process is simple, the fermentation cycle is short, the operation is simple, the equipment requirement is low, the cost is low, and the product is suitable for industrial production.

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Abstract

一种罗伊氏乳杆菌SS23-52及其干粉发酵剂的制备与在纯种益生菌酸奶中的应用。罗伊氏乳杆菌SS23-52在中国微生物菌种保藏管理委员会普通微生物中心的保藏编号为CGMCC No.15152。

Description

[根据细则37.2由ISA制定的发明名称]　一种罗伊氏乳杆菌SS23-52及其干粉发酵剂的制备方法与在纯种益生菌酸奶中的应用 技术领域
本发明涉及一种空间罗伊氏乳杆菌SS23-52及其干粉发酵剂的制备与在纯种益生菌酸奶中的应用。
背景技术
罗伊氏乳杆菌(Lactobacillus reuteri)细胞形态从短杆状、长短不等的长杆状到丝状，单生或短链状排列，革兰氏阳性耐氧或微好氧菌，于MRS培养基平板上菌落大小为1～2mm、表面光滑湿润、边缘不规则、扁平、半透明、灰白色，可进行异型乳酸发酵，能发酵葡萄糖产生乳酸、乙酸、乙醇和CO 2。该菌常栖息于人和动物的肠道内，在生长代谢过程中能产生胞外多糖，使其对肠黏膜具有很强的黏附能力，拮抗肠道致病菌的定殖，可调节肠道菌群平衡，改善肠道健康，对缓解由于肠道菌群失衡导致的小儿便秘具有很好效果。此外，该菌在生长代谢过程中能利用甘油产生一种特殊的抑菌物质--罗伊氏素(reuterin)，其主要成分是3-羟基丙醛(3-HPA)，属于非蛋白质类广谱抗菌物质，能广泛抑制埃希氏菌、志贺氏菌、沙门氏菌、李斯特菌、弧菌、梭菌、葡萄球菌、幽门螺旋杆菌等胃肠道致病菌的生长，避免罹患肠道疾病，对预防和治疗小儿腹泻效果较好。
空间微生物由于受到太空微重力效应、高真空、极端温差、弱磁场和高能粒子(电子、质子、重离子)辐射等诱变作用，可显著提高突变频率而发生基因突变，将导致其生物学性状(如个体形态、菌落特征、生理生化特性、免疫原性等)、发酵生产性能(如生物量、产物量、酶活力、效价、发酵速度等)发生改变。利用天宫2号和神舟11号宇宙飞船搭载返回地面经太空诱变后的罗伊氏乳杆菌，以地面原始的罗伊氏乳杆菌作对照，选育出发生正向突变的发酵性能优良的菌株，再利用遗传稳定且发酵牛乳性能优良的空间罗伊氏乳杆菌优化高密度发酵条件和冻干保护剂配方，制备高活力干粉发酵剂，再应用干粉发酵剂研发生产纯种益生菌酸奶。目前国内外学者对空间微生物的研究主要集中在空间病原菌、空间腐蚀菌和微生物制药方面，而且国内学者对罗伊氏乳杆菌酸奶的研究，也仅仅限于在嗜热链球菌和德氏乳杆菌保加利亚亚种基础上添加罗伊氏乳杆菌研制复合益生菌酸奶，其生理功效显然不如纯种罗伊氏乳杆菌酸奶。因此，本发明对空间罗伊氏乳杆菌的选育及其在纯种益生菌酸奶中的应用，填补了空间食品微生物工程菌的研究空白。故而，有关空间罗伊氏乳杆菌及其纯种益生菌酸奶的发酵生产方法，尚未见国内外发明专利和文献报道。
发明公开
本发明的目的是提供一种空间罗伊氏乳杆菌SS23-52及其干粉发酵剂的制备与在纯种益生菌酸奶中的应用。
本发明首先提供了一种可用于制备纯种益生菌酸奶的菌株—罗伊氏乳杆菌(Lactobacillus reuteri)Fullarton-H-SS23-52，该菌株可简称为空间罗伊氏乳杆菌SS23-52，其在中国微生物菌种保藏管理委员会普通微生物中心的保藏编号为CGMCC No.15152。
本发明还提供了一种菌剂，其活性成分为罗伊氏乳杆菌(Lactobacillus reuteri)Fullarton-H-SS23-52。
所述菌剂为将罗伊氏乳杆菌(Lactobacillus reuteri)Fullarton-H-SS23-52进行培养得到的培养物。
所述菌剂具体可为将罗伊氏乳杆菌(Lactobacillus reuteri)Fullarton-H-SS23-52接种于MRS液体培养基进行培养得到的培养物。
所述菌剂中，所述罗伊氏乳杆菌的活菌数量具体可为7.5×10 9CFU/mL。
所述菌剂还可以包括载体。所述载体可为固体载体或液体载体。所述固体载体可为糖醇类、蛋白类或维生素类物质；所述糖醇类载体可为海澡糖、乳糖、蔗糖、麦芽糊精、麦芽糖、蔗糖、果糖、甘露醇和山梨醇中的至少一种；所述蛋白类载体为脱脂乳粉、乳清粉、酵母粉和酪蛋白的至少一种；所述维生素类载体可为维生素C和/或维生素E。所述液体载体可为甘油、植物油或水。所述菌剂中，所述活性成分可以以培养的活细胞、活细胞的发酵液、细胞培养物的滤液或细胞与滤液的混合物的形式存在。所述组合物的剂型可为多种剂型，如液剂、乳剂、悬浮剂、粉剂、颗粒剂、可湿性粉剂或水分散粒剂。
以上任一所述菌剂的用途可为制备酸奶。
本发明还保护一种干粉发酵剂，是将罗伊氏乳杆菌(Lactobacillus reuteri)Fullarton-H-SS23-52或其发酵产物与冻干保护剂混合后冻干得到的。
所述发酵产物具体可为发酵后的活菌体。
每1g所述干粉发酵剂中，所述罗伊氏乳杆菌(Lactobacillus reuteri)Fullarton-H-SS23-52的活菌数量为如下(a1)或(a2)：
(a1)1.0～5.0×10 10CFU；(a2)4.4×10 10CFU。
所述冻干保护剂中含有如下(b1)或(b2)成分：
(b1)5g/100mL～20g/100mL麦芽糊精和5g/100mL～20g/100mL脱脂奶粉；
(b2)5g/100mL麦芽糊精和10g/100mL脱脂奶粉。
所述冻干保护剂由5g/100mL～20g/100mL麦芽糊精、5g/100mL～20g/100mL脱脂奶粉和溶剂组成；所述溶剂为水，更具体为蒸馏水。
所述冻干保护剂由5g/100mL麦芽糊精、10g/100mL脱脂奶粉和溶剂组成；所述溶剂为水，更具体为蒸馏水。
所述冻干保护剂经过高压灭菌(具体可通过0.07Mpa高压蒸汽灭菌15min实现)。
所述罗伊氏乳杆菌(Lactobacillus reuteri)Fullarton-H-SS23-52的发酵产物为将罗伊氏乳杆菌(Lactobacillus reuteri)Fullarton-H-SS23-52在液体 MRS培养基中进行发酵得到的发酵产物。
所述发酵产物具体可为发酵后的活菌体。
所述发酵的体系中，所述罗伊氏乳杆菌(Lactobacillus reuteri)Fullarton-H-SS23-52的含量为下述(d1)或(d1)或(d1)：
(d1)(0.8～3.8)×10 8CFU/mL；
(d2)(1.5～3.0)×10 8CFU/mL；
(d3)1.5×10 8CFU/mL或2.3×10 8CFU/mL。
所述罗伊氏乳杆菌(Lactobacillus reuteri)Fullarton-H-SS23-52可存在于以上任一所述菌剂中，通过向液体MRS培养基中接种所述菌剂达到引入罗伊氏乳杆菌(Lactobacillus reuteri)Fullarton-H-SS23-52的目的。
在所述菌剂中，所述罗伊氏乳杆菌(Lactobacillus reuteri)Fullarton-H-SS23-52的活菌数量为7.5×10 9CFU/mL时，在所述发酵的体系中，所述菌剂的接种量可为(d4)或(d5)或(d6)：
(d4)1.0％～5.0％(体积百分浓度)，即所述发酵的体系中所述罗伊氏乳杆菌(Lactobacillus reuteri)Fullarton-H-SS23-52的活菌数为(0.8～3.8)×10 8CFU/mL；
(d5)2.0％～4.0％(体积百分浓度)，即所述发酵的体系中所述罗伊氏乳杆菌(Lactobacillus reuteri)Fullarton-H-SS23-52的活菌数为(1.5～8.0)×10 8CFU/mL；
(d6)2.0％或3.0％(体积百分浓度)，即所述发酵的体系中所述罗伊氏乳杆菌(Lactobacillus reuteri)Fullarton-H-SS23-52的活菌数为1.5×10 8CFU/mL或2.3×10 8CFU/mL。
所述发酵的温度为(e1)或(e2)或(e3)：
(e1)31℃～43℃；(e2)34℃～40℃；(e3)37℃。
所述发酵的时间为(f1)或(f2)或(f3)：
(f1)8小时～24小时；(f2)12小时～20小时；(f3)16小时或20小时。
所述发酵过程中，保持发酵体系的pH＝6.8(具体可通过流加20g/100mL的NaOH溶液实现)。
所述发酵的搅拌转速具体可为120～150r/min。
以上任一所述发酵产物具体可为将所述发酵后的发酵体系离心后得到的沉淀(菌泥)。所述离心条件具体可为4℃、4000r/min离心20min。
所述发酵的活菌体沉淀产物和所述冻干保护剂的混合比例具体可为：每100mL发酵体系离心后得到的活菌体沉淀产物加入10mL冻干保护剂均匀混合。
所述冻干具体可为将发酵产物和冻干保护剂的混合物于-80℃预冻2～4h至完全冻结状态，然后于-55℃、真空度0.13mBar的条件下冻干36～48h至完全干燥状态。
所述干粉发酵剂的用途可为制备酸奶。
本发明还保护罗伊氏乳杆菌(Lactobacillus reuteri)Fullarton-H-SS23-52，或，以上任一所述菌剂，或，以上任一所述干粉发酵剂在制备酸奶中的应用。
本发明还保护一种酸奶的制备方法，包括如下步骤：向原料乳中添加罗伊氏乳杆菌(Lactobacillus reuteri)Fullarton-H-SS23-52，或，以上任一所述菌剂，或，以上任一所述干粉发酵剂，进行发酵，得到酸奶。
所述发酵体系中，所述罗伊氏乳杆菌(Lactobacillus reuteri)Fullarton-H-SS23-52的含量为下述(c1)或(c2)或(c3)：
(c1)(2.2～13.0)×10 7CFU/mL；
(c2)(4.4～11.0)×10 7CFU/mL；
(c3)4.4×10 7CFU/mL。
上述方法中，所述向原料乳中添加罗伊氏乳杆菌(Lactobacillus reuteri)Fullarton-H-SS23-52可通过向所述原料乳中添加以上任一所述干粉发酵剂实现。
在所述干粉发酵剂中，所述罗伊氏乳杆菌(Lactobacillus reuteri)Fullarton-H-SS23-52的活菌数量为4.4×10 10CFU/g时，在所述发酵的体系中，所述干粉发酵剂的接种量可为(c4)或(c5)或(c6)：
(c4)0.5‰～3.0‰(质量体积千分浓度)，即所述发酵的体系中所述罗伊氏乳杆菌(Lactobacillus reuteri)Fullarton-H-SS23-52的活菌数为(2.2～13.0)×10 7CFU/mL；
(c5)1.0‰～2.5‰(质量体积千分浓度)，即所述发酵的体系中所述罗伊氏乳杆菌(Lactobacillus reuteri)Fullarton-H-SS23-52的活菌数为(4.4～11.0)×10 7CFU/mL；
(c6)1.0‰(质量体积千分浓度)，即所述发酵的体系中所述罗伊氏乳杆菌(Lactobacillus reuteri)Fullarton-H-SS23-52的活菌数为4.4×10 7CFU/mL。
所述发酵体系中，还包括绵白糖或蔗糖。
所述酸奶的制备方法具体可为：将原料乳(具体可为利乐枕包装，净含量240mL/袋，内蒙古伊利实业集团股份有限公司)加热至60℃，加入5.5g/100mL绵白糖(蔗糖质量含量为95％)，继续加热至90℃保温5～10min，冷却至37℃，得到灭菌原料乳，以1.0‰(质量体积千分浓度)的接种量将以上任一所述罗伊氏乳杆菌(Lactobacillus reuteri)Fullarton-H-SS23-52干粉发酵剂接入，搅拌均匀，于37℃发酵至凝固，得到酸奶。
所述酸奶含有所述罗伊氏乳杆菌(Lactobacillus reuteri)Fullarton-H-SS23-52的活菌数量为3.2×10 9CFU/mL，酸度为54.71°T，凝乳时间为3.5h，突破了利用嗜热链球菌和德氏乳杆菌保加利亚亚种两种菌的共生作用生产商品化普通酸奶的凝乳时间(发酵时间为5～6h)。
以上任一所述原料乳为哺乳动物乳汁或由所述哺乳动物乳汁得到的液体乳制品，可用来制备酸奶。所述原料乳具体可为牛乳。
本发明还保护以上任一所述方法制备得到的酸奶。
实际生产中，采用罗伊氏乳杆菌(Lactobacillus reuteri)Fullarton-H-SS23-52及其干粉发酵剂制备酸奶的工艺流程如下：
1、原料乳：原料乳选用纯牛乳(细菌的总菌数一般低于10 4CFU/mL，不含抗生素和消毒药，不宜选用患乳房炎乳)。
2、净化：原料乳以离心机除去牛乳中的白细胞和其他肉眼可见的杂质。
3、标准化：原料乳的主要成分指标应符合食品卫生国家标准GB 5408-85。其总干物质应不低于11.5％，脂肪含量根据产品不同大体调整为4种：3.2％、2.5％、1.0％和<0.1％，可通过脱除奶油或添加1％～3％的脱脂奶粉或稀奶油以调整总干物质或脂肪含量。
4、预热、配料和过滤：将原料乳加热至60℃左右，加入5.5g/100mL的绵白糖(蔗糖质量含量为95％)，溶解后过滤除杂。
5、均质：在均质机中于8～10MPa压力下对预热的原料乳进行均质处理。目的是使乳凝固均匀，质地更加细腻、平滑，亦可使脂肪球变小而防止脂肪上浮。
6、杀菌：将原料乳加热至90℃，保温5～10min。
7、冷却：经杀菌后的原料乳迅速冷却至37℃，待接种。
8、接种：将空间罗伊氏乳杆菌SS23-52干粉发酵剂(空间罗伊氏乳杆菌SS23-52的活菌数为4.4×10 10CFU/g)接种于原料乳中，接种量为1.0‰(质量体积千分浓度，即发酵体系中空间罗伊氏乳杆菌SS23-52的活菌数为4.4×10 7CFU/mL)。
9、分装：为避免杂菌侵入，分装于塑料小容器的操作应在无菌室中快速进行。
10、保温发酵：将塑料小容器置于发酵室中保持发酵温度37℃。当发酵乳的酸度达到55～70°T，乳凝固性状良好时，即发酵成熟。发酵时间为3～4h。
11、冷却：将盛酸奶的容器从发酵室取出，用冷风迅速冷却至10℃以下。
12、冷藏与后熟：经冷却处理的酸奶，贮藏于0～5℃冷藏室中保存，直至饮用。
以上任一酸奶具体可为益生菌酸奶，更具体可为罗伊氏乳杆菌纯种益生菌酸奶。
本发明利用天宫2号和神舟11号宇宙飞船搭载返回地面经太空诱变后的罗伊氏乳杆菌，以地面原始的罗伊氏乳杆菌作对照，选育出发生正向突变的发酵性能优良的菌株空间罗伊氏乳杆菌SS23-52，再通过优化高密度发酵条件和冻干保护剂，研发了一种利用该菌株制备的干粉发酵剂的方法，可用于研发生产纯种益生菌酸奶。利用本发明的空间罗伊氏乳杆菌SS23-52得到的纯种益生菌酸 奶，有浓郁的炒麦风味，口感细腻和润滑，酸甜度适中，酸度为54.71°T，凝乳结实，乳清析出较少，凝乳时间缩短至3.5h，突破了利用嗜热链球菌和德氏乳杆菌保加利亚亚种两种菌的共生作用生产商品化普通酸奶的凝乳时间(发酵时间为5～6h)。
生物材料保藏说明
生物材料的分类命名：罗伊氏乳杆菌(Lactobacillus reuteri)
生物材料的菌株编号：Fullarton-H-SS23-52
生物材料的保藏单位名称：中国微生物菌种保藏管理委员会普通微生物中心
生物材料的保藏单位简称：CGMCC
生物材料的保藏单位地址：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，邮政编码：100101
生物材料的保藏日期：2018年01月02日
生物材料的保藏中心登记入册编号：CGMCC No.15152。
附图说明
图1为空间罗伊氏乳杆菌(Lactobacillus reuteri)SS23-52生产纯种益生菌酸奶的流程工艺。
实施发明的最佳方式
以下的实施例便于更好地理解本发明，但并不限定本发明。下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的试验材料，如无特殊说明，均为自常规生化试剂商店购买得到的。以下实施例中的定量试验，均设置三次重复实验，结果取平均值。
地面罗伊氏乳杆菌(Lactobacillusreuteri)GS23：美国模式菌种收集中心(American type culture collection)，编号：ATCC23272。
下述实施例中的MRS液体培养基由溶质和溶剂组成，溶剂为蒸馏水，溶质及其浓度分别为：酪蛋白胨10g/L、牛肉膏10g/L、酵母粉5g/L、葡萄糖10g/L、乙酸钠5g/L、柠檬酸二铵2g/L、吐温-80 1mL/L、K 2HPO 42g/L、MgSO 4·7H 2O 0.2g/L、MnSO 4·H 2O 0.05g/L，pH6.8。MRS固体培养基为向MRS液体培养基中加入琼脂粉1.7g/L得到的培养基。
实施例1、空间罗伊氏乳杆菌菌株的筛选
一、菌株的太空诱变
将地面罗伊氏乳杆菌(Lactobacillusreuteri)GS23经天宫二号和神州十一号飞船搭载返回，得到太空诱变菌种，从中分离纯化得到115株菌株，分别标记菌株代号为SS23-1至SS23-115。
二、菌株活化
将冻存于-80℃冰柜的地面罗伊氏乳杆菌(Lactobacillusreuteri)GS23和 菌株SS23-1至SS23-115甘油保藏管分别按2％～3％接入5mL MRS液体培养基中，37℃培养16h，连续三代活化后用于后续试验。
三、菌株的初筛
将步骤二得到的地面罗伊氏乳杆菌(Lactobacillusreuteri)GS23和菌株SS23-1至SS23-115的液体MRS培养物，按2％的接种量接入5mL脱脂乳试管中，置于37℃培养至乳凝固后，记录各菌株(F1代)凝乳时间，并描述凝乳情况。再如此连续操作3代(F2-F4)，记录每代的凝乳时间和凝乳情况(如表1所示)，从中筛选出凝乳时间较短(发酵牛乳速度较快)，凝乳结实，乳清析出较少的发酵优良菌株。
表1 空间罗伊氏乳杆菌凝乳时间和凝乳状态结果
Figure PCTCN2018094621-appb-000001
Figure PCTCN2018094621-appb-000002
Figure PCTCN2018094621-appb-000003
Figure PCTCN2018094621-appb-000004
由表1可见，地面罗伊氏乳杆菌(Lactobacillusreuteri)GS23于脱脂乳试管中凝乳时间较长，一般需要2～3d使牛乳凝固。只有SS23-12、SS23-22、SS23-24、SS23-27、SS23-30、SS23-36、SS23-38、SS23-46、SS23-52、SS23-84、SS23-88菌株的凝乳时间明显缩短，其中SS23-12、SS23-27、SS23-36、SS23-38、SS23-46、SS23-52、SS23-84、SS23-88空间罗伊氏乳杆菌的凝乳时间缩短至7h，且凝乳结实，说明该8株空间罗伊氏乳杆菌在太空环境条件下相关基因发生了正突变。
四、菌株的复筛
按2％接种量将上述筛选的SS23-12、SS23-27、SS23-36、SS23-38、SS23-46、SS23-52、SS23-84、SS23-88菌株脱脂乳试管纯粹培养物以2％(体积百分数)接种量移入盛有100mL灭菌脱脂乳的三角瓶中，37℃培养至牛乳凝固后进行菌 种发酵剂活力测定，包括凝乳时间、酸度、pH、活菌计数，从中选取发酵性能优良的菌株，制备酸奶(酸奶发酵条件：发酵剂接种量3％，绵白糖用量5g/100mL、37℃)记录酸奶凝固时间，并按表2和表3对酸奶品质进行感官评价。空间罗伊氏乳杆菌发酵剂活力测定结果和发酵剂制作酸奶感官评价结果见表4和表5。
表2 酸奶品质评分标准
Figure PCTCN2018094621-appb-000005
表3 酸奶质量标准
Figure PCTCN2018094621-appb-000006
表4 空间罗伊氏乳杆菌发酵剂活力测定结果
Figure PCTCN2018094621-appb-000007
由表4可见，利用SS23-36、SS23-84、SS23-88菌株制作发酵剂的凝乳时间均为11～12h，酸奶凝乳时间为7h；而SS23-12、SS23-27、SS23-38、SS23-46、SS23-52菌株发酵剂凝乳时间均为7h，酸奶凝乳时间为3～4h，且酸度和活菌数均较高。结论：SS23-12、SS23-27、SS23-38、SS23-46、SS23-52菌株为发酵速度最快、产酸较高的菌株。
表5 空间罗伊氏乳杆菌发酵剂制作酸奶感官评价结果
Figure PCTCN2018094621-appb-000008
Figure PCTCN2018094621-appb-000009
由表5可见，SS23-27菌株制作酸奶的感官评分最高，其次是SS23-88、SS23-52、SS23-46、SS23-12、SS23-38菌株，有浓郁的炒麦风味，酸甜度适中，口感细腻和润滑，凝乳结实，乳清析出较少。因此，空间SS23-27、SS23-88、SS23-52、SS23-46、SS23-12、SS23-38菌株为发酵牛乳性能优良的菌株。
五、菌株的遗传稳定性检测
将上述复筛得到的发酵性能优良的SS23-12、SS23-27、SS23-36、SS23-38、SS23-46、SS23-52、SS23-84、SS23-88菌株，于5mL液体MRS培养基连续传代50代(接种量2％～3％，37℃培养16h)，而后按2％的接种量接入5mL脱脂乳试管中，于37℃培养至乳凝固后，记录凝乳时间和凝乳状态。在牛乳中如此连续操作6代，50代传代的空间罗伊氏乳杆菌凝乳时间和凝乳状态结果如表6所示。
表6 50代传代的空间罗伊氏乳杆菌凝乳时间和凝乳状态结果
Figure PCTCN2018094621-appb-000010
由表6可见，经50代传代后，SS23-52菌株在牛乳中经6次发酵验证，F3代后凝乳时间均保持在7h，显示其发酵性能遗传最稳定。而SS23-12、SS23-36、SS23-38、SS23-46、SS23-84、SS23-88菌株的凝乳发酵性能稳定性有所降低。
结论：SS23-52菌株为遗传稳定性最好，且发酵牛乳性能最好的菌株，可成为开发功能性发酵乳制品的最有潜力菌株。
六、SS23-52菌株的形态学鉴定与分子学鉴定
将SS23-52菌株进行革兰氏染色，结果显示SS23-52菌株为革兰氏阳性菌。形态学观察，SS23-52菌株的菌体呈短杆状、长短不等的长杆状到丝状，单生或短链状排列；于MRS培养基平板上菌落大小为1～2mm、表面光滑湿润、边缘不规则、扁平、半透明、灰白色。
检测SS23-52菌株的16S rDNA，测序结果如序列表的序列1所示。16s rDNA鉴定结果显示SS23-52菌株与罗伊氏乳杆菌Lactobacillus reuteri的相似性达到100％。经过形态学及16S rDNA鉴定，可以确定SS23-52菌株属于罗伊氏乳 杆菌。
七、SS23-52菌株的保藏
将SS23-52菌株命名为罗伊氏乳杆菌(Lactobacillus reuteri)Fullarton-H-SS23-52，于2018年1月2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)，保藏编号为CGMCC NO.15152。罗伊氏乳杆菌(Lactobacillus reuteri)Fullarton-SS23-52简称为空间罗伊氏乳杆菌SS23-52。
实施例2、空间罗伊氏乳杆菌SS23-52高密度发酵条件的优化
一、空间罗伊氏乳杆菌SS23-52发酵剂的制备
将空间罗伊氏乳杆菌SS23-52甘油保藏管分别按2％～3％(体积百分浓度)接入5mL MRS液体培养基中，37℃静置培养16h活化，活化后的MRS试管培养物按2％(体积百分浓度)接种量移入盛有200mL灭菌MRS培养基的三角瓶中，37℃静置培养16h，得到空间罗伊氏乳杆菌SS23-52发酵剂，检测发酵剂中罗伊氏乳杆菌的活菌数量为7.5×10 9CFU/mL。
所述空间罗伊氏乳杆菌SS23-52发酵剂的检测方法：取1mL发酵剂放入含有99mL灭菌生理盐水中，采用拍击式匀质器以8000～10000r/min的速度处理2min，充分振荡后，制成10 -2的均匀稀释液。再以9mL灭菌生理盐水做10倍递增稀释至10 -8，取10 -6～10 -8的稀释液各1mL置于无菌平皿中，倒入溶化并冷却至46℃的MRS固体培养基约15mL，迅速轻轻旋动平皿，使培养基与菌液充分混匀，每个稀释度3次重复。同时将MRS固体培养基注入加有1mL无菌生理盐水的灭菌平皿内作空白对照。待培养基凝固后，翻转平板，置(36±1)℃温箱中培养(48±2)h，待菌落长出后即可计数。
二、单因素多水平试验优化空间罗伊氏乳杆菌SS23-52高密度发酵条件
1、发酵温度的确定
将步骤一制备的空间罗伊氏乳杆菌SS23-52发酵剂按照3％(体积百分浓度)接种量接种于200mLMRS液体培养基中，发酵体系初始pH为6.8，将发酵体系在不同发酵温度(31℃、34℃、37℃、40℃、43℃)下发酵16h，对发酵液中的活菌浓度进行检测。采用MRS琼脂培养基以倾注平板培养法检测发酵液中的活菌数量，每个稀释度设3次重复。空间罗伊氏乳杆菌SS23-52不同发酵温度的发酵液中活菌数结果如表7所示。
表7 空间罗伊氏乳杆菌SS23-52不同发酵温度的发酵液中活菌数结果
Figure PCTCN2018094621-appb-000011
温度是影响高密度发酵液中生物产量的重要因素。由表7可知，在发酵温度为31～37℃之间时，发酵液活菌数逐渐升高，而在发酵温度在37～43℃之间时，发酵液活菌数逐渐下降。说明空间罗伊氏乳杆菌SS23-52的较适宜发酵温度在34～40℃之间，其中温度为37℃时，发酵液活菌数最高，为8.57×10 9CFU/mL。故确定空间罗伊氏乳杆菌SS23-52发酵温度为37℃。
2、发酵接种量的确定
将步骤一制备的空间罗伊氏乳杆菌SS23-52发酵剂按照不同接种量(1％、2％、3％、4％、5％，体积百分浓度)接种于200mLMRS液体培养基中，发酵体系初始pH为6.8，将发酵体系在37℃下发酵16h，对发酵液中的活菌浓度进行检测。采用MRS琼脂培养基以倾注平板培养法检测发酵液中的活菌数量，每个稀释度设3次重复。空间罗伊氏乳杆菌SS23-52不同接种量的发酵液中活菌数结果如表8所示。
表8 空间罗伊氏乳杆菌SS23-52不同接种量的发酵液中活菌数结果
Figure PCTCN2018094621-appb-000012
接种量的大小决定于生产菌种在发酵罐中的繁殖速度和生物产量。若接种量过低，菌体繁殖速度缓慢；如接种量过高，菌体消耗培养基中的营养物质过快，造成后期生物产量降低。由表8可知，在接种量为1％～2％之间时，发酵液活菌数逐渐升高，而在发酵温度在2％～5％之间时，发酵液活菌数逐渐下降。说明空间罗伊氏乳杆菌SS23-52的较适宜接种量在2％～3％之间，其中接种量为2％时，发酵液活菌数最高，为7.72×10 9CFU/mL。故确定空间罗伊氏乳杆菌SS23-52发酵接种量为2％(体积百分浓度，发酵剂中空间罗伊氏乳杆菌SS23-52的活菌数为7.5×10 9CFU/mL，即发酵体系中空间罗伊氏乳杆菌SS23-52的活菌数为1.5×10 8CFU/mL)。
3、发酵时间的确定
将步骤一制备的空间罗伊氏乳杆菌SS23-52发酵剂按照2％(体积百分浓度)接种量接种于200mLMRS液体培养基中，发酵体系初始pH为6.8，将发酵体系在37℃下发酵不同时间(8h、12h、16h、20h、24h)，对发酵液中的活菌浓度进行检测。采用MRS琼脂培养基以倾注平板培养法检测发酵液中的活菌数量，每个稀释度设3次重复。空间罗伊氏乳杆菌SS23-52不同发酵时间的发酵液中活菌数结果如表9所示。
表9 空间罗伊氏乳杆菌SS23-52不同发酵时间的发酵液中活菌数结果
Figure PCTCN2018094621-appb-000013
发酵时间的长短直接影响发酵液中菌体的生物产量。如发酵时间过短，菌体尚未达到生命力极为旺盛的对数生长期，而对数生长期的末期菌体生物量才达到最高峰，故较短的发酵时间会造成生物产量降低；如发酵时间过长，菌体生长进入衰亡期，出现菌体自溶导致生物量下降。由表9可知，在发酵时间为8～16h之间时，发酵液活菌数逐渐升高，而在发酵温度在20～24h之间时，发酵液活菌数逐渐下降。说明空间罗伊氏乳杆菌SS23-52的较适宜发酵时间在16～20h之间，其中发酵时间为20h时，发酵液活菌数最高，为7.53×10 9CFU/mL。故确定空间罗伊氏乳杆菌SS23-52发酵时间为20h。
综上所述，通过单因素多水平试验，确定空间罗伊氏乳杆菌SS23-52高密度发酵条件为：发酵温度为37℃，接种量为2％(体积百分浓度，发酵剂中空间罗伊氏乳杆菌SS23-52的活菌数为7.5×10 9CFU/mL，即发酵体系中空间罗伊氏乳杆菌SS23-52的活菌数为1.5×10 8CFU/mL)，发酵时间为20h。
三、正交试验优化空间罗伊氏乳杆菌SS23-52高密度发酵条件
在步骤二的基础上，设计发酵温度、发酵剂接种量、发酵时间三因素三水平[L 9(3 4)]正交试验(见表10)，在5L全自动发酵罐中，以2L液体MRS为发酵培养基，在分批发酵过程中控制发酵体系pH6.8(具体通过流加20g/100mL的NaOH溶液控制pH)，搅拌转速为120～150r/min，采用MRS琼脂培养基以倾注平板培养法检测发酵液中的活菌数量，每个稀释度设3次重复，通过对试验结果的极差分析和K值分析确定较优的高密度发酵工艺条件。正交试验优化菌株SS23-52高密度发酵条件结果如表11所示。
表10 三因素三水平[L 9(3 4)]正交试验表
Figure PCTCN2018094621-appb-000014
表11 正交试验[L 9(3 4)]优化菌株SS23-52高密度发酵条件结果
Figure PCTCN2018094621-appb-000015
Figure PCTCN2018094621-appb-000016
注：表中数据均为n＝3之测定平均值。
由表11可见，根据正交试验极差分析可知，不同发酵条件对发酵液活菌数的影响顺序为：发酵温度＞发酵时间＞接种量；根据正交试验K值分析KA2＞KA3＞KA1、KB2＞KB3＞KB1、KC2＞KC3＞KC1可知，菌株SS23-52高密度发酵条件的最优组合为A2B2C2，即发酵温度37℃、发酵剂接种量3％(体积百分浓度)、发酵时间16h。
综上所述，本试验研究罗伊氏乳杆菌高密度发酵条件，主要从发酵液活菌数指标来考虑。经极差分析和K值析，确定空间罗伊氏乳杆菌SS23-52高密度发酵条件组合是A2B2C2，即发酵温度为37℃、发酵剂接种量为3％(体积百分浓度，发酵剂中空间罗伊氏乳杆菌SS23-52的活菌数为7.5×10 9CFU/mL，即发酵体系中空间罗伊氏乳杆菌SS23-52的活菌数为2.3×10 8CFU/mL)、发酵时间为16h，同时控制发酵体系pH6.8，搅拌转速为120～150r/min。
四、空间罗伊氏乳杆菌SS23-52高密度发酵正交试验验证试验
在上述正交试验得出的优化发酵条件基础上，将空间罗伊氏乳杆菌SS23-52发酵剂按3％接种量移入2L液体MRS培养基中，于37℃搅拌发酵16h，其他发酵条件与正交试验相同，同时以对照条件(发酵温度37℃、接种量2％、发酵时间20h)为对照组，测定发酵液中的活菌数量，结果如表12所示。
表12 发酵条件优化结果与初始发酵条件结果对照
Figure PCTCN2018094621-appb-000017
注：表中数据均为n＝3之测定平均值。
由表12可见，在优化发酵条件下菌株SS23-52发酵液中的活菌数为1.6×10 10CFU/mL，而对照组发酵液中的活菌数为9.5×10 9CFU/mL。因此，在优化发酵条件下，菌株SS23-52高密度发酵液中的活菌数是优化前的1.68倍。
实施例3、空间罗伊氏乳杆菌SS23-52干粉发酵剂的制备
一、离心浓缩与优化保护剂配方
1、将实施例2的步骤一制备的空间罗伊氏乳杆菌SS23-52发酵剂按3％(体积百分浓度，发酵剂中空间罗伊氏乳杆菌SS23-52的活菌数为7.5×10 9CFU/mL，即发酵体系中空间罗伊氏乳杆菌SS23-52的活菌数为2.3×10 8CFU/mL)接种量接 种于含有2.4L液体MRS培养基的5L全自动发酵罐中，控制发酵温度37℃、发酵体系pH6.8、搅拌转速为120～150r/min、发酵时间16h，得到发酵液。
2、按照表13所示，以蒸馏水配制8种不同组合的30mL冻干保护剂，于0.07Mpa高压蒸汽灭菌15min。
3、将步骤1发酵结束后的发酵液均分为8份后于4℃、4000r/min离心20min，弃上清液，收集菌泥，分别与上述8种30mL无菌冻干保护剂均匀混合(实际应用中，可将每100mL发酵体系离心后得到的菌泥加入10mL冻干保护剂均匀混合)，得到浓缩活菌制剂。采用MRS培养基以倾注平板培养法检测浓缩活菌制剂的活菌数量，每个稀释度设3次重复，结果见表13，以确定冻干保护剂较优组合配方。
冻干前的浓缩活菌制剂活菌数量的检测方法：称取1mL浓缩活菌制剂放入含有99mL灭菌生理盐水中，采用拍击式匀质器以8000～10000r/min的速度处理3min，充分振荡后，制成10 -2的均匀稀释液；再以9mL灭菌生理盐水做10倍递增稀释至10 -9，取10 -7～10 -9的稀释液各1mL置于无菌平皿中，倒入溶化并冷却至46℃的MRS琼脂培养基约15mL，迅速轻轻旋动平皿，使培养基与菌液充分混匀，每个稀释度3次重复。同时将MRS琼脂培养基注入加有1mL无菌生理盐水的灭菌平皿内作空白对照。待培养基凝固后，翻转平板，置(36±1)℃温箱中培养(48±2)h，待菌落长出后即可计数。
二、预冻与冻干
将步骤一制备的8中浓缩活菌制剂于-80℃预冻2～4h至完全冻结状态，得到预冻活菌制剂。利用6L LABCONCO真空冷冻干燥机(美国)将预冻活菌制剂于-55℃、真空度0.13mBar的条件下，冻干36～48h至完全干燥状态，得到冻干活菌制剂。采用MRS培养基以倾注平板培养法检测冻干后的制剂活菌数量，计算菌种存活率，每个稀释度设3次重复，结果见表13。
冻干后的活菌制剂活菌数量的检测方法：称取1g冻干活菌制剂，加入定量的灭菌生理盐水(按30mL浓缩活菌制剂经冻干后得到冻干活菌制剂的质量，计算加入灭菌生理盐水的用量，制成还原浓缩活菌制剂)，采用漩涡振荡器振荡1min后，得到均匀的浓缩活菌制剂；取1mL浓缩活菌制剂，放入含有99mL灭菌生理盐水中，采用拍击式匀质器以8000～10000r/min的速度处理3min，充分振荡后，制成10 -2的均匀稀释液；再以9mL灭菌生理盐水做10倍递增稀释至10 -9，取10 -7～10 -9的稀释液各1mL置于无菌平皿中，倒入溶化并冷却至46℃的MRS琼脂培养基约15mL，迅速轻轻旋动平皿，使培养基与菌液充分混匀，每个稀释度3次重复。同时将MRS琼脂培养基注入加有1mL无菌生理盐水的灭菌平皿内作空白对照。待培养基凝固后，翻转平板，置(36±1)℃温箱中培养(48±2)h，待菌落长出后即可计数。
表13 不同组合的冻干保护剂对空间罗伊氏乳杆菌SS23-52存活率和活菌数量的影响
Figure PCTCN2018094621-appb-000018
Figure PCTCN2018094621-appb-000019
由表13可见，4号冻干保护剂组合的空间罗伊氏乳杆菌SS23-52存活率最高，为98.67％；8号冻干保护剂组合的菌种存活率次之，为97.67％；1号、2号、3号、5号冻干保护剂组合菌种存活率在91.49～95.86之间，6号和7号冻干保护剂组合菌种存活率最小，分别为86.33％和86.90％。故确定空间罗伊氏乳杆菌SS23-52的冻干保护剂较优组合配方为：5g/100mL麦芽糊精+10g/100mL脱脂奶粉，其菌种存活率可达98％以上，冻干发酵剂的活菌数4.4×10 10CFU/g。
综上所述，采用单因素多水平试验优化空间罗伊氏乳杆菌SS23-52冻干保护剂的较优组合配方为：5g/100mL麦芽糊精+10g/100mL脱脂奶粉，其菌种存活率可达98％以上，冻干发酵剂的活菌数4.4×10 10CFU/g。
实施例4、空间罗伊氏乳杆菌SS23-52干粉发酵剂在纯种益生菌酸奶中的应用
1、将实施例2的步骤一制备的空间罗伊氏乳杆菌SS23-52发酵剂按3％(体积百分浓度，发酵剂中空间罗伊氏乳杆菌SS23-52的活菌数为7.5×10 9CFU/mL，即发酵体系中空间罗伊氏乳杆菌SS23-52的活菌数为2.3×10 8CFU/mL)接种量接种于含有2L液体MRS培养基的5L全自动发酵罐中，控制发酵温度37℃、发酵体系pH6.8、搅拌转速为120～150r/min、发酵时间16h，得到发酵液。
2、完成步骤1后，将发酵液以4℃、4000r/min离心20min，弃上清液，收集菌泥。
3、配制冻干保护剂200mL，冻干保护剂为含有5g/100mL麦芽糊精和10g/100mL脱脂奶粉的水溶液，于0.07Mpa高压蒸汽灭菌15min。
4、将步骤2制备的菌泥与步骤3制备的冻干保护剂均匀混合，于-80℃预冻2～4h至完全冻结状态，得到预冻活菌制剂。利用6L LABCONCO真空冷冻干燥机(美国)将预冻活菌制剂于-55℃、真空度0.13mBar的条件下，冻干36～48h至完全干燥状态，得到空间罗伊氏乳杆菌SS23-52干粉发酵剂(空间罗伊氏乳杆菌SS23-52干粉发酵剂中空间罗伊氏乳杆菌SS23-52的活菌数为4.4×10 10CFU/g)。
5、将纯牛奶(利乐枕包装，净含量240mL/袋，内蒙古伊利实业集团股份有限公司)加热至60℃，加入5.5g/100mL绵白糖(蔗糖质量含量为95％)，继续加热至90℃保温5～10min，冷却至37℃，得到灭菌牛乳；分别以0.5‰、1.0‰、1.5‰、2.0‰、2.5‰、3.0‰(质量体积千分浓度)的接种量将空间罗伊氏乳杆菌SS23-52干粉发酵剂接入上述灭菌牛乳中，搅拌均匀，于37℃发酵至牛乳凝固，记录凝乳时间，测定酸奶4℃后熟后(置于4℃，12h)的酸度、pH和活菌数量，并对成品益生菌酸奶按表14和表15内容进行感官评价，结果如表16所示。
表14 酸奶品质评分标准
Figure PCTCN2018094621-appb-000020
表15 酸奶质量标准
Figure PCTCN2018094621-appb-000021
表16 空间罗伊氏乳杆菌SS23-52干粉发酵剂不同接种量的酸奶综合指标结果
Figure PCTCN2018094621-appb-000022
由表16可见，当空间罗伊氏乳杆菌SS23-52干粉发酵剂的接种量为0.5‰～3.0‰时，酸奶在4h之内完全凝乳，牛乳凝固结实，乳清析出较少，且随着SS23-52干粉发酵剂接种量的增加，凝乳时间越短，由4.00h缩短至2.50h；但随着SS23-52干粉发酵剂的接种量的增加，酸奶的酸度逐渐升高，使得酸奶的酸甜度逐渐不适中，导致酸奶的感官评价分数逐渐降低，但其中以接种量为1.0‰的干粉发酵剂制备酸奶的感官评价分数最高，为58.58分，次之是1.5‰和2.0‰的 接种量，感官评价分数分别为58.58和57.55。故确定制备纯种益生菌酸奶添加空间罗伊氏乳杆菌SS23-52干粉发酵剂的最佳用量为1.0‰。
终上所述，以直投式空间罗伊氏乳杆菌SS23-52干粉发酵剂制备纯种益生菌酸奶的发酵工艺条件：发酵温度为37℃、干粉发酵剂添加量为1.0‰(质量体积千分浓度，空间罗伊氏乳杆菌SS23-52干粉发酵剂中罗伊氏乳杆菌SS23-52的活菌数量为4.4×10 10CFU/g，即发酵体系中空间罗伊氏乳杆菌SS23-52的活菌数为4.4×10 7CFU/mL)、凝乳时间为3.5h。成品酸奶有浓郁的炒麦风味，口感细腻和润滑，酸甜度适中，凝乳结实，乳清析出较少，活菌数量为3.2×10 9CFU/mL，酸度为54.71°T。突破了利用嗜热链球菌和德氏乳杆菌保加利亚亚种两种菌的共生作用生产商品化普通酸奶的凝乳时间(发酵时间为5～6h)。
利用空间罗伊氏乳杆菌(Lactobacillus reuteri)SS23-52干粉发酵剂生产纯种益生菌酸奶的工艺流程见图1，具体如下：
1、原料乳：原料乳选用纯牛乳(细菌的总菌数一般低于10 4CFU/mL，不含抗生素和消毒药，不宜选用患乳房炎乳)。
2、净化：原料乳以离心机除去牛乳中的白细胞和其他肉眼可见的杂质。
3、标准化：原料乳的主要成分指标应符合食品卫生国家标准GB 5408-85。其总干物质应不低于11.5％，脂肪含量根据产品不同大体调整为4种：3.2％、2.5％、1.0％和<0.1％，可通过脱除奶油或添加1％～3％的脱脂奶粉或稀奶油以调整总干物质或脂肪含量。
4、预热、配料和过滤：将原料乳加热至60℃左右，加入5.5g/100mL的绵白糖(蔗糖质量含量为95％)，溶解后过滤除杂。
5、均质：在均质机中于8～10MPa压力下对预热的原料乳进行均质处理。目的是使乳凝固均匀，质地更加细腻、平滑，亦可使脂肪球变小而防止脂肪上浮。
6、杀菌：将原料乳加热至90℃，保温5～10min。
7、冷却：经杀菌后的原料乳迅速冷却至37℃，待接种。
8、接种：将空间罗伊氏乳杆菌SS23-52干粉发酵剂(空间罗伊氏乳杆菌SS23-52的活菌数为4.4×10 10CFU/g)接种原料乳中，接种量为1.0‰(质量体积千分浓度，空间罗伊氏乳杆菌SS23-52干粉发酵剂中罗伊氏乳杆菌SS23-52的活菌数量为4.4×10 10CFU/g，即发酵体系中空间罗伊氏乳杆菌SS23-52的活菌数为4.4×10 7CFU/mL)。
9、分装：为避免杂菌侵入，分装于塑料小容器的操作应在无菌室中快速进行。
10、保温发酵：将塑料小容器置于发酵室中保持发酵温度37℃。当发酵乳的酸度达到55～70°T，乳凝固性状良好时，即发酵成熟。发酵时间为3～4h。
11、冷却：将盛酸奶的容器从发酵室取出，用冷风迅速冷却至10℃以下。
12、冷藏与后熟：经冷却处理的酸奶，贮藏于0～5℃冷藏室中保存，直至饮用。
目的是防止酸奶继续发酵产酸造成pH过低而影响口感，并防止杂菌污染繁殖。冷藏条件下的后熟有利于酸奶风味物质的形成，最终获得有浓郁的炒麦风味，口感细腻和润滑，酸甜度适中，凝乳结实，乳清析出较少的纯种益生菌成品酸奶。
工业应用
本发明对空间罗伊氏乳杆菌的选育及其在纯种益生菌酸奶中的应用，填补了空间食品微生物工程菌的研究空白。另外，本发明制作纯种益生菌酸奶的原料来源方便，发酵工艺简单，发酵周期短，操作简单，对设备要求低，成本较低，适于工业化生产。

Claims (14)

  1. 罗伊氏乳杆菌(Lactobacillus reuteri)Fullarton-H-SS23-52，其在中国微生物菌种保藏管理委员会普通微生物中心的保藏编号为CGMCC No.15152。
  2. 一种菌剂，其活性成分为权利要求1所述的罗伊氏乳杆菌(Lactobacillus reuteri)Fullarton-H-SS23-52。
  3. 一种干粉发酵剂，是将权利要求1所述的罗伊氏乳杆菌(Lactobacillus reuteri)Fullarton-H-SS23-52或其发酵产物与冻干保护剂混合后冻干得到的。
  4. 如权利要求3所述的干粉发酵剂，其特征在于：所述罗伊氏乳杆菌(Lactobacillus reuteri)Fullarton-H-SS23-52的发酵产物为将权利要求1所述的罗伊氏乳杆菌(Lactobacillus reuteri)Fullarton-H-SS23-52在液体MRS培养基中进行发酵得到的发酵产物。
  5. 如权利要求3或4所述的干粉发酵剂，其特征在于：每1g所述干粉发酵剂中，所述罗伊氏乳杆菌(Lactobacillus reuteri)Fullarton-H-SS23-52的活菌数量为如下(a1)或(a2)：
    (a1)1.0～5.0×10 10CFU；(a2)4.4×10 10CFU。
  6. 如权利要求3至5任一所述的干粉发酵剂，其特征在于：
    所述冻干保护剂中含有如下(b1)或(b2)成分：
    (b1)5g/100mL～20g/100mL麦芽糊精和5g/100mL～20g/100mL脱脂奶粉；
    (b2)5g/100mL麦芽糊精和10g/100mL脱脂奶粉。
  7. 权利要求1所述的罗伊氏乳杆菌(Lactobacillus reuteri)Fullarton-H-SS23-52在制备酸奶中的应用。
  8. 权利要求2所述的菌剂在制备酸奶中的应用。
  9. 权利要求3至6任一所述的干粉发酵剂在制备酸奶中的应用。
  10. 一种酸奶的制备方法，包括如下步骤：向原料乳中添加权利要求1所述的罗伊氏乳杆菌(Lactobacillus reuteri)Fullarton-H-SS23-52，进行发酵，得到酸奶。
  11. 一种酸奶的制备方法，包括如下步骤：向原料乳中添加权利要求2所述的菌剂，进行发酵，得到酸奶。
  12. 一种酸奶的制备方法，包括如下步骤：向原料乳中添加权利要求3至6任一所述的干粉发酵剂，进行发酵，得到酸奶。
  13. 如权利要求10-12任一所述的方法，其特征在于：所述发酵体系中，罗伊氏乳杆菌(Lactobacillus reuteri)Fullarton-H-SS23-52的含量为下述(c1)或(c2)或(c3)：
    (c1)(2.2～13.0)×10 7CFU/mL；(c2)(4.4～11.0)×10 7CFU/mL；
    (c3)4.4×10 7CFU/mL。
  14. 权利要求10-13任一所述的方法制备得到的酸奶。
PCT/CN2018/094621 2018-02-23 2018-07-05 一种罗伊氏乳杆菌ss23-52及其干粉发酵剂的制备方法与在纯种益生菌酸奶中的应用 WO2019161631A1 (zh)

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