Disclosure of Invention
In view of the above, the invention aims to provide a lactococcus lactis IMAU11823 capable of producing a sticky cheese-producing flavor and application thereof.
In order to achieve the purpose, the invention provides the following technical scheme: a lactococcus lactis IMAU11823 capable of producing cheese-like aroma is preserved in China general microbiological culture Collection center with the preservation number of CGMCC No. 16243.
Preferably, the lactococcus lactis IMAU11823 has the morphological characteristics that cells are ellipsoid-shaped, 6.2-7.3 micrometers in length and 4.4-5.1 micrometers in width and appear in pairs or short chains.
The application further aims to provide application of the high-yield casein-aroma-producing lactococcus lactis IMAU11823 in preparation of fermented foods.
Preferably, the food is a fermented dairy product or a health product.
Preferably, when the food is a health product, the food further comprises a dietetic carrier or an additive support, and the food is in the form of powder, granules, tablets, capsules, suspensions, emulsions, syrups, liquids, extracts, teas, jellies or beverages.
Preferably, when the food product is a fermented dairy product, the food product further comprises one or more of gelatin, pectin, hydroxypropyl distarch phosphate and agar.
Preferably, the food further comprises a leavening agent, wherein the leavening agent is one or a combination of more of lactobacillus bulgaricus, streptococcus thermophilus, lactobacillus acidophilus, bifidobacterium, lactobacillus casei and lactobacillus rhamnosus.
It is another object of the present application to provide a dairy starter and its use for dairy fermentation, said dairy starter containing an effective content of said lactococcus lactis IMAU11823 and an acceptable carrier. According to the specific implementation mode of the application, the lactococcus lactis IMAU11823 provided by the application can be compounded with a proper amount of other conventional leavening agents to prepare a compound leavening agent for fermenting dairy products. Preferably, the conventional leavening agent comprises one or more of lactobacillus bulgaricus, streptococcus thermophilus, lactobacillus acidophilus, bifidobacterium, lactobacillus casei, lactobacillus rhamnosus and the like.
Particularly, when the conventional leavening agent is a combination of the above-mentioned strains, the amount ratio of each strain can be reasonably adjusted and selected by one skilled in the art on the premise of smooth reaction.
It is another object of the present application to provide a fermented dairy product fermented with said lactococcus lactis IMAU11823 or said dairy starter.
It is another object of the present application to provide a method for fermenting milk using said lactococcus lactis IMAU11823, said method comprising: adding the lactococcus lactis IMAU11823 into sterilized dairy products, and carrying out oxygen isolation and heat preservation reaction, wherein the dairy products are soybean milk and/or cow milk.
Optionally, the addition amount of the lactococcus lactis IMAU11823 is 0.5X 106CFU/mL~3×106CFU/mL, preferably 1X 106CFU/mL~2×106CFU/mL; the temperature of the heat preservation fermentation is (30 +/-0.5) DEG C; the time for heat preservation and fermentation is 8-12 h, preferably (9.5-10.5) h.
In an achievable manner, the method of fermenting milk using the lactococcus lactis IMAU11823 may be: adding lactococcus lactis IMAU11823 into sterilized milk, wherein the inoculation amount is 1 × 106CFU/mL; shaking up and shaking for 30-50 s to ensure that the lactococcus lactis is uniformly dispersed in the dairy product, so that the growth and fermentation of thalli are facilitated; the mixed milk product system after shaking up is placed in a constant temperature incubator at 30 ℃, and the incubation at 30 ℃ is beneficial to the rapid growth and propagation of thalli, so that the time cost can be saved. The lactococcus lactis IMAU11823 was in optimal growth under the above conditions.
The invention has the beneficial effects that: the Lactococcus lactis IMAU11823 capable of producing acid by adhesion can rapidly ferment cow milk; the milk can be sticky in a large amount in the milk fermentation process, and the fermented milk product is endowed with unique taste; meanwhile, the fermented dairy product can produce cheese fragrance and endow the fermented dairy product with unique flavor.
Biological preservation Instructions
The Lactococcus lactis (Lactococcus lactis) IMAU11823 provided by the invention is preserved in the China Committee for culture Collection of microorganisms and strains, and the preservation number is as follows: CGMCC No.16243, preservation date of 2018, 8 and 9 months, and preservation address of No. 3 Hospital No.1 of West Lu of Beijing, Chaoyang, North Chen, China.
Detailed Description
The invention provides a lactococcus lactis IMAU11823 with high viscosity and flavor yield, which is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No. 16243. In the invention, the lactococcus lactis IMAU11823 has the characteristics of high fermentation speed, high viscosity and fermentation for producing a cheese flavor, and has good proteolysis capability. The morphological characteristics of the lactococcus lactis IMAU11823 disclosed by the invention are as follows: the cells are in an ellipsoid shape, have the length of 6.2-7.3 mu m and the width of 4.4-5.1 mu m, and appear in pairs or short chains; an opalescent colony is grown on an M17 culture medium, is opaque and circular, has a smooth surface and a convex center, and has the diameter of about 1.2-1.5 mm. The lactococcus lactis IMAU11823 originally comes from the traditional fermented food chewing in Mongolian cylinder Haote city in China.
The invention also provides application of the lactococcus lactis IMAU11823 in preparation of fermented food. In the invention, the food is a fermented dairy product or a health product; the fermented dairy product preferably comprises yogurt, kefir, fermented buttermilk, fermented milks, kefir, chews, and the like. The fermented dairy product of the invention preferably also comprises auxiliary materials; the auxiliary materials preferably comprise one or more of gelatin, pectin, hydroxypropyl distarch phosphate and agar. The application of the invention is preferably to obtain the fermented dairy product by fermenting with lactococcus lactis IMAU11823 as a fermentation strain. The method for obtaining the fermented milk product by fermentation in the invention comprises the following steps: inoculating activated lactococcus lactis IMAU11823 into skim milk, and performing oxygen isolation, heat preservation and fermentation. The activated lactococcus lactis is preferably M17 culture solution of lactococcus lactis, wherein the concentration of lactococcus lactis is preferably (1-10). times.106cfu/ml; the ratio of lactococcus lactis to skim milk is excellentSelected from (1-10) × 106cfu: 100g of the total weight of the mixture; the oxygen-insulating heat-preserving fermentation temperature is preferably 29.5-30.5 ℃, more preferably 30 ℃, and the fermentation time is preferably 7-10 hours, more preferably 8 hours.
The lactococcus lactis IMAU11823 provided by the invention can be compounded with a proper amount of other conventional leavening agents to prepare a compound leavening agent for fermenting dairy products. Preferably, the conventional leavening agent comprises one or more of lactobacillus bulgaricus, streptococcus thermophilus, lactobacillus acidophilus, bifidobacterium, lactobacillus casei, lactobacillus rhamnosus and the like. The proportion relationship of the components in the leavening agent is not particularly limited in the invention, as long as fermented milk products can be realized.
The composite leaven containing lactococcus lactis IMAU11823 provided by the invention is applied to preparing fermented dairy products, can be rapidly fermented, and has strong acid and viscosity producing capability and good protein hydrolysis performance.
The invention also provides a health food comprising the lactococcus lactis IMAU 11823. The health food preferably further comprises a dietetically acceptable carrier or additive. The formulation of the health food in the present invention is preferably powder, granule, tablet, capsule, suspension, emulsion, syrup and extract. The health food is preferably jelly or beverage; the jelly is preferably yogurt jelly; the beverage preferably comprises a yoghurt drink.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
The sources of the tested strains in the following experimental examples of the application are respectively:
lactococcus lactis IMAU11823 was prepared as in example 1;
the leavening agent A is purchased from Italian dairy research center CSL;
the starter B was purchased from Kohansen (China) GmbH.
EXAMPLE 1 isolation, purification and characterization of Lactococcus lactis (Lactococcus lactis) IMAU11823
Lactococcus lactis (Lactococcus lactis) IMAU11823 was isolated from a chewing gum of a traditional fermented food in the domestic Haaste city of Mongolian cylinder, specifically, isolated by a TPY selective medium, and expanded by selecting a single strain.
Lactococcus lactis (Lactococcus lactis) is inoculated into an M17 liquid medium, cultured for 24 hours at 37 ℃, subcultured for 3 generations, and the strain activity is recovered. Diluting the bacterial liquid by 10 times to obtain 10-1~10-7A dilution gradient. Suck 200. mu.L of 10 cells each-4、10-5、10-6、10-7The diluted solution is evenly spread in a M17 solid medium plate and is subjected to anaerobic culture at 37 ℃ for 24 h. Selecting monoclonals with different shapes, sizes and colors, inoculating the monoclonals into a liquid culture medium, and culturing the monoclonals in a constant-temperature anaerobic incubator at 37 ℃ for 24 hours. After the strain grows well, gram staining and microscopic examination are carried out. Isolates were preserved and strain genomic DNA was extracted for subsequent assay analysis.
Wherein the composition of the M17 culture medium is as follows: 5g of soybean peptone, 2.5g of casein peptone, 5g of beef extract powder, 2.5g of yeast extract powder, 5g of lactose, 0.5g of sodium ascorbate, 19g of beta-sodium glycerophosphate and 0.25g of magnesium sulfate, adjusting the pH to 7.2, adjusting the agar to 12.75g, sterilizing at 121 ℃ for 15 min.
b. Molecular biological identification
Inoculating a frozen and preserved test strain into a TPY enrichment liquid culture medium, culturing at a constant temperature of 30 ℃ for 24h, subculturing for 2-3 generations by TPY, placing 2mL of thallus culture at the last logarithmic growth stage into a sterile EP tube for centrifugation, collecting thallus after centrifugation for 3min (4 ℃) at 8000 Xg, removing supernatant, and extracting the genomic DNA of the strain by adopting a CTAB freeze-thawing method special for lactobacillus.
Wherein, the TPY enrichment culture solution comprises the following components: 10g of lactose, 5g of beef extract, 5g of yeast powder, 10g of casein peptone, 5g of soybean peptone, 2.5g of dipotassium hydrogen phosphate, 2.5g of potassium dihydrogen phosphate, 0.1g of magnesium sulfate, 800.25g of tween, 0.5g of L-cysteine hydrochloride, 15g of agar and 1L of distilled water, sterilizing at 121 ℃ for 15 min.
The 16S rDNA sequence of Lactococcus lactis (Lactococcus lactis) IMAU11823 was sequenced by a Sanger sequencing method, and phylogenetic trees were constructed with the 16S rDNA sequences of Lactococcus lactis 4 strain model strains and Lactococcus pisci model strains. Lactococcus lactis (Lactococcus lactis) IMAU11823 is closely related to Lactococcus lactis subsp.
c. Morphological characteristics
Lactococcus lactis (Lactococcus lactis) IMAU11823 of the present application has the following morphological characteristics: the cells were ellipsoidal, 6.2-7.3 μm long and 4.4-5.1 μm wide, and appeared in pairs, or in short chains (see FIG. 2).
d. Morphological characteristics of colonies
Lactococcus lactis (Lactococcus lactis) IMAU11823 of the present application grown on M17 medium to form an opalescent colony that is opaque, round, smooth in surface, convex in the center, and approximately 1.2-1.5mm in diameter (see FIG. 3).
EXAMPLE 2 fermentation Performance study of Lactococcus lactis (Lactococcus lactis) IMAU11823 fermented milk was prepared from the Lactococcus lactis (Lactococcus lactis) IMAU11823 obtained after the screening, and the fermented milk was prepared using a culture solution of Lactococcus lactis M17 (1X 10) which was activated for the third generation6cfu/ml) was inoculated into 100g of skim milk and left to ferment at 30 ℃ for 8h to obtain the fermented milk IMAU 11823.
Determination of pH in the fermentation of Lactococcus lactis (Lactococcus lactis) IMAU11823. MAU11823, IMAU11866 and IMAU11875 are all isolated from chewing lactococcus lactis, and the results show that the pH values of the three strains are different and obvious after fermentation for 8 hours. Wherein the acid production rate of the lactococcus lactis IMAU11823 fermented cow milk is higher, so that the fermented cow milk reaches the fermentation end point first. The results show that the time taken for the lactococcus lactis IMAU11823 to ferment the cow milk to reach the fermentation end point is the shortest.
TABLE 1 determination of pH and titrated acidity of Lactococcus lactis (Lactococcus lactis) fermentation
Viscosity and water holding capacity measurement of Lactococcus lactis (Lactococcus lactis) IMAU11823 fermented milk
Fermentation experiments were performed on Lactococcus lactis (Lactococcus lactis) IMAU11823, A and B, respectively, and the viscosity and water holding capacity of 3 Lactococcus lactis were measured. Viscosity was measured using a viscometer. About 20g of yogurt was weighed and allowed to stand for 2 hours, and then filtered using a funnel, and the whey mass was weighed out. The ratio of the whey mass to the yogurt mass before filtration is the water holding capacity of the strain. The results are shown in fig. 4, and the lactic acid bacteria (Lactococcus lactis) IMAU11823 fermented yogurt has good viscosity and water holding capacity, which are both significantly higher than those of the other two lactic acid bacteria.
Electronic tongue assay for Lactococcus lactis (Lactococcus lactis) IMAU11823 fermented cow milk
3 strains of lactococcus lactis were fermented for 24 hours, and then flavor substances were measured using an electronic tongue. The results are shown in FIG. 5, which shows that Lactococcus lactis (Lactococcus lactis) IMAU11823 had no noticeable bitter or pungent taste compared with the other two strains.
Sensory evaluation of Lactococcus lactis (Lactococcus lactis) IMAU11823 fermented milk
After fermenting yogurt with 3 strains of Lactococcus lactis (Lactococcus lactis), the fermented soybean milk was subjected to sensory evaluation by 20 sensory evaluators, and the evaluation criteria were as shown in table 2 below. The results are shown in fig. 6 and show that the sensory evaluation of Lactococcus lactis (Lactococcus lactis) IMAU11823 and fermented yogurt is significantly higher than that of the remaining two strains. IMAU11823 scored up to 82 points.
TABLE 2 sensory evaluation index
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.