CN114231456B - Lactobacillus rhamnosus strain RSF-1 and application thereof in dairy cake production - Google Patents

Abstract

The invention relates to the technical field of microorganisms, in particular to a Lactobacillus rhamnosus strain RSF-1 and application thereof in dairy cake production, wherein the Lactobacillus rhamnosus strain RSF-1 has the characteristic of fast acid reduction, and a product obtained by fermenting whey of the strain is used for preparing buffalo milk dairy cakes, the fermented product can provide acid for raw materials, and also can generate various enzymes, so that the taste of the dairy cakes is effectively improved, through inspection, the content of free fatty acid in the whey obtained by fermenting the strain is 100.34 mu mol/L, and other strains in the same period are only 20-80 mu mol/L, so that the strain has stronger capability of decomposing fat and can bring more flavors; the yield of the milk cakes is more than 32 percent, and the yield of other strains is only about 22 to 25 percent; meanwhile, the cheese prepared by fermenting whey by using the strain is strong in milk fragrance, fresh and sweet in taste, soft and tender and good in taste, and has no sour taste or other bad flavors.

Description

Lactobacillus rhamnosus strain RSF-1 and application thereof in dairy cake production

[ technical field ] A

The invention relates to the technical field of microorganisms, in particular to a lactobacillus rhamnosus strain RSF-1 and application thereof in dairy cake production.

[ background ] A method for producing a semiconductor device

The milk cake is a traditional milk product in Yunnan of China, has been for over 600 years to date, and is prepared by heating and sterilizing raw milk, adding a milk coagulant (acid) into the milk, squeezing and molding by utilizing the principle that milk protein can be coagulated and precipitated when meeting acid; in the preparation of the milk cake, a milk coagulant is particularly important, and at present, the common milk coagulant comprises chemically edible acid, such as acetic acid and the like, and also comprises some vegetable protease with acidity, such as: papaya, fig, safflower, ginger juice, thistle plants and the like; the use of these acids, while allowing good coagulation of milk proteins, still exists: naturally fermented whey is easily polluted by infectious microbes, the quality is unstable, the quality of the cheese is influenced, and defects of microbial toxins, even brown color, hard texture and the like can occur. For example, the phenomenon that the quality of the milk cakes is different under different processes is disclosed in the text of 'optimization of milk cake processing process and analysis of nutrient components thereof' in the prior art.

However, the buffalo milk is not the preferred raw material for preparing the milk cake due to low milk protein content, but the buffalo milk is widely cultured in the Guangxi region, and more buffalo milk products need to be developed, and a related preparation method for preparing the milk cake by using the buffalo milk is disclosed in the research on the functional buffalo milk cake preparation process in the prior art, but the research shows that the yield of the milk cake is not high and is only about 22%, and the acid used in the method is acetic acid, so that the milk cake is easily over-hard due to improper operation, and is not suitable for industrial production.

Therefore, a coagulant suitable for buffalo milk raw materials is necessarily developed aiming at buffalo milk, and through continuous research and screening of the applicant, the single lactic acid bacteria can be used for replacing conventional naturally fermented whey, the lactic acid bacteria can generate acid during fermentation and can also form various enzymes, the yield of the milk cake can be effectively high, the taste of the milk cake can be effectively improved, microbial toxin pollution possibly caused by natural fermentation can be effectively avoided, the single bacterial strain fermentation is simple and controllable in process, and the method is suitable for industrial production.

[ summary of the invention ]

In view of the above, there is a need to develop a coagulant suitable for buffalo milk raw material per se, and through continuous research by the applicant, it has been found that by using a single lactic acid bacterium instead of conventional naturally fermented whey, the lactic acid bacterium can not only produce acid during fermentation, but also form various enzymes, thereby effectively increasing the yield of the cheese, effectively increasing the content of free fatty acids, improving the taste of the cheese, increasing the yield of the cheese, and avoiding biotoxin pollution of naturally fermented whey, and being suitable for industrial production.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows:

the microbial agent comprises a strain Lactobacillus rhamnosus RSF-1, and the preservation number is CGMCC NO. 23907: china general microbiological culture preservation management center, address: the microbial research institute of China academy of sciences No. 3, Xilu No. 1, Beijing, Chaoyang, and the preservation date is 11 months and 15 days in 2021.

The invention also comprises a microbial inoculum containing the Lactobacillus rhamnosus RSF-1 strain.

The invention also comprises the application of the Lactobacillus rhamnosus RSF-1 and/or the microbial inoculum containing the Lactobacillus rhamnosus RSF-1 in preparing the milk cakes.

Furthermore, the raw material for preparing the milk cake is buffalo milk.

The invention also comprises the application of the Lactobacillus rhamnosus RSF-1 and/or the microbial inoculum containing the Lactobacillus rhamnosus RSF-1 in improving the free fatty acid content of the dairy cake.

The invention also provides a method for preparing a milk cake by using the Lactobacillus rhamnosus RSF-1 strain and/or the microbial inoculum containing the Lactobacillus rhamnosus RSF-1 strain, which is characterized by comprising the following steps:

(1) culturing a lactobacillus rhamnosus RSF-1 strain;

(2) whey fermentation: taking cow milk whey, sterilizing, inoculating the RSF-1 strain obtained in the step (1) according to the inoculation amount of 5% of the volume of fermentation liquor, and fermenting to obtain fermented whey;

(3) preparing raw materials: taking buffalo raw milk, filtering to remove impurities, adjusting the proportion of protein and fat in the raw milk to ensure that the total solid content of the raw milk is more than or equal to 17, and obtaining the raw milk;

(4) homogenizing raw milk: heating and homogenizing the raw milk obtained in the step (3) for later use;

(5) preheating and sterilizing raw milk: preheating the homogenized raw milk obtained in the step (4) at 90 ℃, and then cooling to 70-75 ℃ for heat preservation for later use;

(6) first addition of fermented whey: uniformly adding the fermented whey obtained in the step (2) into the raw milk in the step (5) at 70 ℃ according to the addition amount of 15-20% (v/w) of the raw milk in the step (5), rapidly stirring, immediately stopping stirring, and keeping the temperature at 70-75 ℃ for 2 min;

(7) adding whey for the second time: adding the fermented whey obtained in the step (2) into the raw milk obtained in the step (6) at 70 ℃ according to 5-10% (v/w) of the raw milk obtained in the step (6), rapidly stirring, immediately stopping stirring, and keeping the temperature at 70-75 ℃ for 2 min;

(8) filtering the curdled milk particles: and (4) continuously coagulating the raw milk obtained in the step (7) at 70 ℃, wherein the coagulation time is 4min, taking out coagulated milk particles, putting the coagulated milk particles into a filtering and squeezing device for pressing and refrigerating.

Further, the pH of the fermented whey of the step (2) is 3.6.

The invention has the following beneficial effects:

the Lactobacillus rhamnosus RSF-1 strain has the characteristic of fast acid reduction, and after the strain is applied to fermenting whey, the whey is used for coagulating protein and fat in raw milk and preparing a milk cake, so that the yield and the taste of the milk cake are effectively improved; according to the invention, the fermented product obtained after whey fermentation is used for preparing the cheese, besides acid can be provided for the raw materials, various enzymes are also generated, the taste of the cheese is effectively improved, through inspection, the content of free fatty acid in the cheese prepared by applying the method disclosed by the invention is 100.64 mu mol/L, and other strains in the same period experiment are only 20-80 mu mol/L; the yield is more than 32 percent, while other strains are only about 22 to 25 percent; meanwhile, the cheese prepared by fermenting whey by using the strain is strong in milk fragrance, fresh and sweet in taste, soft and tender in cheese without sour taste and other bad flavors, and good in taste, and is a novel strain for preparing buffalo milk cheese.

[ description of the drawings ]

FIG. 1 is a diagram showing the morphology of the RSF-1 strain of the present invention on a plate;

FIG. 2 is a microscopic image of the RSF-1 strain of the present invention;

FIG. 3 is a phylogenetic tree of the RSF-1 strain and the model strain of the present invention;

FIG. 4 is a schematic diagram of the finished products of buffalo milk cakes and blackflower buffalo milk cakes fermented by RSF-1 strains according to the invention; in the figure, the raw material of the milk cake A is buffalo raw milk; the raw material of the B milk cake is raw milk of black and white cow.

[ detailed description ] embodiments

All of the features disclosed in this specification, or all of the steps in any method or process so disclosed, may be combined in any combination, except combinations of features and/or steps that are mutually exclusive.

Any feature disclosed in this specification (including any accompanying claims, abstract) is merely an example of a generic series of equivalent or similar features, unless explicitly described as such.

Example 1:

screening of the strain Lactobacillus rhamnosus RSF-1:

lactobacillus rhamnosus strain RSF-1, the preservation number of which is CGMCC NO.23907 is preserved in: china general microbiological culture preservation management center, address: the microbial research institute of China academy of sciences No. 3, Xilu No. 1, Beijing, Chaoyang, and the preservation date is 11 months and 15 days in 2021.

The strain is obtained by naturally fermenting and separating buffalo milk whey in 2021, wherein the buffalo milk whey is from a farm of the local farm and is located in south-ning-Guangxi city, the strain is obtained by separating the buffalo milk whey by adopting an MRS culture medium plate coating method and an inclined plane scribing method, and finally the strain RSF-1 is screened out.

The microorganisms of this example were morphologically classified and identified by molecular biology as follows:

1. morphological Classification of strains

Morphological classification of strains:

inoculating the strain RSF-1 on an MRS culture medium, observing the morphological characteristics of a bacterial colony, wherein the bacterial colony is shown on a plate as shown in figure 1, is white and circular, and has a smooth and wet surface; as shown in FIG. 2, the strain was rod-shaped and nonfilamentous.

2. Molecular biological identification

Sequencing and verifying the strains, and comparing the strains with a corresponding database to construct a phylogenetic tree diagram of the strains: the result is shown in FIG. 3, the strain RSF-1 and the Lactobacillus rhamnosus can be clustered together and belong to the genus Bacillus sp, and the shape is considered to be closer to the species Lactobacillus rhamnosus through morphological identification, so the strain RSF-1 is named as Lactobacillus rhamnosus.

Example 2:

whey fermentation experiment:

the strains screened from the same batch were: inoculating lactobacillus P7, lactobacillus T6 and lactobacillus RSF-1 into buffalo whey according to the inoculation amount of 5%, culturing and fermenting at 37 deg.C for 48h, and measuring pH value and free fatty acid content of whey fermentation;

wherein the pH value is determined by adopting the following steps: a Sedoles pH tester; the content of free fatty acid is measured by adopting a Nanjing constructed free fatty acid detection kit method; the results obtained are shown in table 1:

TABLE 1 buffalo milk whey fermentation results for different strains

Strain numbering	pH value	Free fatty acid (μmol/L)
			P7	3.61	61.85
T6	4.87	38.69
			RSF-1	3.60	100.34

As can be seen from Table 1, the strain RSF-1 of the present application has a high acid-reducing speed of fermented buffalo milk, the pH value is close to that of the P7 strain, but the content of free fatty acid is highest.

Example 3:

as can be seen from example 2, after the strain RSF-1 ferments buffalo whey, the buffalo whey has a lower pH value, the lactobacillus with the low pH value can inhibit most of the growth of mixed bacteria to form a good dominant flora, and has good bacteriostatic activity, and meanwhile, the fermented whey also has a higher content of free fatty acid, so that the strain RSF-1 is selected for preparing the buffalo milk cake, and the specific method is as follows:

(1) strain culture: culturing lactobacillus RSF-1 in sterilized MRS broth at 37 deg.C for 24 hr;

(2) whey fermentation: taking cow milk whey, sterilizing at the pH value of about 6.2 at 95 ℃ for 5min, cooling to 37 ℃, inoculating the RSF-1 lactobacillus in the step (1) according to 3-5% of the volume of fermentation liquor, and fermenting at 37 ℃ for 48h until the pH value of the whey is 3.6; obtaining fermented whey;

(3) preparing raw materials: taking raw buffalo milk, filtering and removing impurities, and adjusting the mass ratio of raw milk protein to fat as follows: 0.65, the total solid content of the raw milk is more than or equal to 17 for standby, and the raw milk is obtained;

(4) homogenizing raw milk: heating the raw milk obtained in the step (3) to 45-50 ℃, homogenizing under the conditions of low pressure of 5mpa and high pressure of 20-25 mpa, and then keeping for later use; (experiments show that the size of milk curds formed in the heat-preservation curding stage after the raw milk is homogenized is uniform, so that the prepared cheese is more uniform and fine, the size of particles formed by the raw milk curds which are not homogenized is different, and the cheese formed by pressing is difficult to be uniform and is easy to form pores);

(5) preheating and sterilizing raw milk: heating the homogenized raw milk obtained in the step (4) to 90 ℃ for preheating, and then cooling to 70-75 ℃ for heat preservation for later use;

(6) first addition of fermented whey: uniformly adding the fermented whey obtained in the step (2) into raw milk at 70 ℃ according to the addition amount of 15-20% (v/w) of the raw milk, rapidly stirring, immediately stopping stirring, and keeping the temperature at 70-75 ℃ for 2 min;

(7) adding whey for the second time: adding the fermented whey obtained in the step (2) into the raw milk in the step (6) according to 5-10% (v/w) of the raw milk, rapidly stirring, immediately stopping stirring, and keeping the temperature at 70-75 ℃ for 2 min;

(8) filtering the curdled milk particles: and (3) continuously coagulating the raw milk obtained in the step (7) at 70 ℃, wherein the coagulation time is 4min, taking out coagulated milk particles, and putting the coagulated milk particles into a filtering and squeezing device, wherein the pressing condition is as follows: the pressure is 0.4mpa, and the pressing time is 2 h;

(9) cooling and shaping: putting the pressed and formed milk cake in the step (8) into a refrigerator at 4 ℃ for more than 6 hours; (the pressed and formed milk cakes need to be refrigerated and shaped, and the actual cases show that the milk cakes with the same raw material quality and the process are more stable after being refrigerated and shaped, the shape of the milk cakes is better kept during vacuum packaging, and the milk cakes which are not refrigerated and shaped are easier to collapse and deform during vacuum packaging);

(10) packaging a finished product: and (4) carrying out vacuum packaging on the cheese shaped in the step (9), and preserving and storing at 4 ℃.

The process is optimized mainly aiming at: the curd temperature in the step (8), the pH value of the whey in the step (2), whether the raw milk in the step (5) is preheated, the number of whey additions and the coagulation time in the step (8); calculating the product yield and measuring the masticatory force of the milk cakes, wherein the calculation formula of the milk cake yield is as follows: the yield of the milk cake is (milk cake mass/total mass of raw materials) multiplied by 100 percent; the chewing force is tested by adopting a TPA texture analyzer, the mass percent of the milk protein of the raw material is 4.88 percent, the mass percent of the fat is 7.52 percent, and the inoculation amount is 5 percent of the volume of the fermentation liquor; specific experimental parameters and results are shown in table 2:

TABLE 2 optimization of the cheese making process

As can be seen from Table 2, according to the calculation of the product yield, the buffalo milk cake obtained by the 2 nd group of processes has the highest yield, the cake yield is 32.5 percent, and the chewing force is the lowest, which indicates that under the condition, the cake yield is the highest, and the cake has the best and tender mouthfeel; the culture conditions for group 2 were: the raw milk is heated to 90 ℃ for preheating, then the temperature is reduced, whey is added, the curd temperature is 70 ℃, the whey pH3.6, the whey adding times are 2 times, and the curd time is 4 min.

The following were obtained by sensory evaluation: the samples at 70 ℃ in groups 1-3 have fine, soft and smooth mouthfeel, strong milk fragrance, good formability and strong cookability (the samples are heated and decocted without breakage);

the curd samples of groups 4-6 at 80 ℃ and groups 7-9 at 90 ℃ had a slightly harder mouthfeel;

and (3) comparing after decocting: the sample of 70 ℃ (group 1-group 3) is tender in mouth, the sample of 80 ℃ (group 4-group 6) and 90 ℃ (group 7-group 9) has stronger bruxism, and the mouth feel is poorer, corresponding to the chewing force, the chewing force of the sample of 70 ℃ (group 1-group 3) is low, and the mouth feel is softer and tender, the sample of 80 ℃ (group 4-group 6) and the sample of 90 ℃ (group 7-group 9) has larger chewing force and poorer mouth feel.

Thus, it is further demonstrated that the buffalo milk cake produced by the process conditions of group 2 has the best effect, yield and mouthfeel.

Meanwhile, in the embodiment, a set of experiments for producing the black and white cow milk cakes by taking the group 2 as a condition are also performed, wherein the raw milk has the fat mass percentage of 3.55 percent and the protein mass percentage of 2.96 percent; the results show that: the black and white cow is used as a milk source to prepare the milk cakes, and the milk cake yield is as follows: 17-18%, and the protein curd particles are smaller in the curd process, which is not beneficial to whey removal, the prepared cheese is softer in quality and poor in formability, and the yield is lower than that of the cheese adopting buffalo milk as a milk source, which indicates that the process and the strain of the application are possibly more suitable for producing the buffalo milk cheese, and the optimal curd temperature of the Holstein milk is 85 ℃ found by the pottery (2017) research, and the curd temperature of the process is lower, so that the milk protein in the Sporotrichum and white flower cows cannot be effectively coagulated.

The results of the finished product prepared specifically are shown in fig. 4: in fig. 4, a is a milk cake prepared according to the conditions of group 2 with buffalo milk as the milk source; b, taking the black and white cow milk as a milk source, and preparing the obtained milk cakes according to the conditions of the group 2; it can be seen in the figure that: the milk cake A is white in appearance, smooth, fine, white and tender, good in color and luster, and smooth and well-shaped in cut surface; the milk cake B is faint yellow in appearance, has rough surface, has more granular substances on the section, and is fragile and not shaped. Therefore, the effect of producing the milk cakes by fermenting buffalo milk by the strain RSF-1 is better than that of the black and white cow, which is probably because the component compositions of two different milk sources are different, and the effect of the milk cakes is finally influenced.

Example 4:

in this embodiment, different strains are used for cheese processing of buffalo milk, specifically as follows:

the 9 quick-acid-producing whey fermentation strains screened by the applicant and the preparation method of the embodiment 3 are adopted to process the buffalo milk cakes, the specific preparation method and the specific process are completely the same, the fermentation time of the strains in whey is 48 hours, and the different strains are different; the free fatty acid content in whey (determined by Nanjing as a free fatty acid assay kit), the yield of the milk cake (calculated according to reference example 3), the degree of proteolysis (measured according to Abdel-Hamid (2019) literature), and the taste results (evaluated according to the evaluation criteria of Table 4) are shown in Table 3, where the milk cake prepared from different strains was tasted by slicing immediately after the preparation:

TABLE 3 milk cake yield and taste score for different strains

From the free fatty acids of the whey fermentation of Table 3, RSF-1 strain was the highest in content, and the remaining strains were slightly lower; from the milk cake yield, the milk cake yield processed by two strains of RSF-1 and P7 can reach more than 27 percent, but the whey free fatty acid content and the protein hydrolysis degree of the P7 strain are not as good as those of the RSF-1 strain, which shows that the protein has fewer decomposed amino acids, less flavor substances and less taste than the RSF-1; the participating tasters also showed that the cake produced by the P7 strain had a greater milky smell, while the cake made by the RSF-1 strain had a strong milky smell, no unpleasant smell, and a higher sensory taste score, while the degree of proteolysis of the RSF-1 strain was close to that of the D3 strain, and the taste score was similar, but the cake yield of the D3 strain was lower than that of the RSF-1 strain, only 22.33%; therefore, compared with other strains, the strain RSF-1 has higher yield and good mouthfeel when used for preparing buffalo milk cakes.

When the evaluation is carried out, 10 evaluation experts in the field respectively carry out tasting and scoring, and the average value of scores is taken as a final score, wherein the scoring standard of sensory scores is shown in table 4:

TABLE 4 sensory evaluation criteria for milk cakes

In conclusion, the Lactobacillus rhamnosus RSF-1 strain can effectively improve the coagulation capacity of protein and fat of buffalo milk and improve the yield of the milk cake of the buffalo milk, and meanwhile, the strain can effectively improve the taste of the milk cake and the content of free fatty acid in whey when being used for preparing the milk cake of the buffalo milk, so that the strain is a good microbial inoculum and coagulant for preparing the milk cake of the buffalo milk.

The above examples are merely illustrative of several embodiments of the present invention, and the description thereof is more specific and detailed, but not to be construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present invention should be subject to the appended claims.

Claims (7)

1. Lactobacillus rhamnosus strain (Lactobacillus rhamnosus) RSF-1, characterized in that the preservation number of the strain is CGMCC NO. 23907.

2. A microbial agent comprising the Lactobacillus rhamnosus strain (Lactobacillus rhamnosus) RSF-1 according to claim 1.

3. Use of a Lactobacillus rhamnosus strain (Lactobacillus rhamnosus) RSF-1 according to claim 1 or a bacterial preparation comprising a Lactobacillus rhamnosus strain RSF-1 according to claim 2 for the preparation of a dairy cake.

4. Use according to claim 3, wherein the raw material for preparing the dairy cake is buffalo milk.

5. Use of a Lactobacillus rhamnosus strain (Lactobacillus rhamnous) RSF-1 according to claim 1 or a bacterial preparation comprising a Lactobacillus rhamnosus strain RSF-1 according to claim 2 for increasing the free fatty acid content of fermented whey.

6. A method for preparing a dairy cake using the Lactobacillus rhamnosus strain (Lactobacillus rhamnosus) RSF-1 according to claim 1 or the microbial inoculum comprising the Lactobacillus rhamnosus strain (Lactobacillus rhamnosus) RSF-1 according to claim 2, characterized in that the method comprises the steps of:

(1) culturing Lactobacillus rhamnosus strain (Lactobacillus rhamnosus) RSF-1;

(2) whey fermentation: taking cow milk whey, adjusting the pH value to 6.2, sterilizing, inoculating the Lactobacillus rhamnosus strain (Lactobacillus rhamnosus) RSF-1 obtained in the step (1) according to the inoculation amount of 3-5% of the volume of fermentation liquor, and fermenting to obtain fermented whey;

(3) preparing raw materials: taking buffalo raw milk, filtering to remove impurities, adjusting the proportion of protein and fat in the raw milk to ensure that the total solid content of the raw milk is more than or equal to 17, and obtaining the raw milk;

(4) homogenizing raw milk: heating and homogenizing the raw milk obtained in the step (3) for later use;

(5) preheating and sterilizing raw milk: preheating the homogenized raw milk obtained in the step (4) at 90 ℃, and then cooling to 70-75 ℃ for heat preservation for later use;

(6) first addition of fermented whey: uniformly adding the fermented whey obtained in the step (2) into the raw milk in the step (5) according to the addition amount of 15-20% (v/w) of the raw milk in the step (5), quickly stirring, immediately stopping stirring, and keeping the temperature at 70-75 ℃ for 2 min;

(7) adding whey for the second time: adding the fermented whey obtained in the step (2) into the raw milk obtained in the step (6) according to 5-10% (v/w) of the raw milk obtained in the step (6), rapidly stirring, immediately stopping stirring, and keeping the temperature at 70-75 ℃ for 2 min;

(8) filtering the curdled milk particles: and (4) continuously coagulating the raw milk obtained in the step (7) at 70 ℃, wherein the coagulation time is 4min, taking out coagulated milk particles, putting the coagulated milk particles into a filtering and squeezing device for pressing and refrigerating.

7. The method according to claim 6, wherein the pH of the fermented whey of step (2) is 3.60.

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