Composite leavening agent, fermented milk and preparation method thereof

Abstract

The present invention relates to a composite starter, fermented milk and a preparation method thereof. The composite starter comprises thermophilic streptococcus CICC 6063 and lactobacillus helveticus CICC 6064, wherein the ratio of the number of live bacteria of the thermophilic streptococcus CICC 6063 to that of the lactobacillus helveticus CICC 6064 is 5-20:1. The strains in the composite starter provided by the present invention have a synergistic symbiotic effect, and the starter obtained by compounding has the characteristics of high number of live bacteria and acid stability after storage period, which can not only promote the acidification fermentation reaction of dairy products, but also shorten the fermentation time, and the prepared fermented milk has a strong milky aroma and a floral and fruity aroma without additives and sucrose, and has a fine texture, a soft taste, and no peculiar smell or foreign body sensation.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial fermentation, and in particular to a composite leavening agent, fermented milk and a preparation method thereof. Background Art

[0002] Fermented milk is a low-pH product made from raw cow (sheep) milk or milk powder through sterilization and inoculation of fermentation agents. Fermentation agents play a vital role in the preparation of fermented milk. Excellent fermentation agents can give fermented milk good flavor and taste. At present, the homogenization of fermented milk in my country is serious. Most low-temperature liquid fermented milk is flavored by adding some new flavors and contents, lacking innovation. However, the research and development of fermentation agents by Chinese dairy companies is still in its infancy, and there is a lack of strains with independent intellectual property rights, which restricts the development of the industry.

[0003] Studies have shown that aldehydes, esters, and ketone compounds are the main substances that promote the formation of yogurt flavor. The flavor of fermented milk is generally considered to be determined by a mixture formed by the balance of multiple important volatile compounds. Among them, 2,3-butanedione (also known as diacetyl), acetoin, 2-butanone, etc. are the main flavor substances of yogurt. A 1:1 ratio of acetaldehyde to diacetyl can produce a typical yogurt flavor. Too high a ratio of acetaldehyde will produce an unwelcome "fruity taste". Acetate compounds are crucial to the development of the fruity characteristics of dairy products. They contribute to the fruity and floral aromas in fermented milk, while reducing the strong odors of fatty acids and amines. The volatile flavor substance citronellol is a natural compound that is used in industrial production for spices and perfumes. Its acute toxicity data is 3450 mg / kg for rats and 2650 mg / kg for rabbits, with a threshold of 40 μg / kg. At present, there has been no report of its detection in fermented milk prepared only by starter without adding other excipients.

[0004] Therefore, it is of great significance to develop a high-quality composite dairy starter to solve the homogenization problem of existing fermented milk and to provide a fermented milk with a unique flavor and a preparation method thereof. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a composite starter, fermented milk and a preparation method thereof. The strains in the composite starter provided by the present invention have a synergistic symbiotic effect, and the starter obtained by compounding has the characteristics of high viable count and acid stability after storage period, which can not only promote the acidification fermentation reaction of dairy products, but also shorten the fermentation time. The prepared fermented milk has a rich milky and floral aroma without additives and sucrose, and has a fine texture, a soft taste, and no peculiar smell or foreign body feeling.

[0006] In a first aspect, the present invention provides a composite leavening agent, comprising thermophilic Streptococcus CICC6063 and Lactobacillus helveticus CICC 6064, wherein the ratio of the number of viable bacteria of the thermophilic Streptococcus CICC 6063 to the number of viable bacteria of the Lactobacillus helveticus CICC 6064 is 1-1000:1, for example, 2:1, 5:1, 10:1, 20:1, 50:1, 100:1, 200:1, 500:1, 800:1, etc.; preferably, 1-100:1, for example, 10:1, 20:1, 40:1, 60:1, 80:1, etc., and more preferably, 5-20:1, for example, 8:1, 10:1, 12:1, 14:1, 16:1, 18:1, etc.

[0007] The present invention selects suitable strains for compounding and utilizes their complex symbiotic mechanism to obtain a composite starter that can not only promote the acidification fermentation reaction of dairy products, shorten the fermentation time, and improve the quality and taste of fermented milk products, but also control the growth of microorganisms and reduce the generation of odors. Specifically:

[0008] The thermophilic streptococcus CICC 6063 provided by the present invention has high heat resistance and strong acidification ability, and can improve the quality, nutrition and taste of fermented milk, while the lactobacillus helveticus CICC 6064 has extremely strong proteolytic ability, adaptability and antibacterial ability. The present invention obtains a composite starter by compounding the thermophilic streptococcus CICC 6063 and the lactobacillus helveticus CICC 6064, has a synergistic symbiotic effect, and has the characteristics of high viable bacteria count, fast fermentation speed and acid stability after storage period. The prepared fermented milk product has many kinds of flavor substances and high content, has unique floral and fruity fragrance and rich milky fragrance, high nutritional value and good taste. It not only has broad application value and promotion space, but also will help to provide more diversified, reliable and nutritional value options for dairy product production and consumption.

[0009] As a preferred technical solution of the present invention, the ratio of the number of live bacteria of the thermophilic streptococcus CICC 6063 to the number of live bacteria of the lactobacillus helveticus CICC 6064 is 5-10:1. When the ratio of the number of live bacteria of the thermophilic streptococcus CICC 6063 to the number of live bacteria of the lactobacillus helveticus CICC 6064 is within this range, the fermentation time can be significantly shortened, the number of live bacteria in the prepared fermented milk is relatively high, and it has an obvious weak post-acid effect, and the content of aldehyde compounds is above 19200 μg / kg, and the content of ethyl ester compounds is above 1250 μg / kg, giving the fermented milk a flower and fruit aroma, especially the ethyl acetate content is above 1150 μg / kg, and the fermented milk also has a pleasant creamy sweetness; the ratio of acetaldehyde to diacetyl (2,3-butanedione) is between 1.09-1.27:1, which can produce a typical yogurt flavor; thereby making the prepared fermented milk have excellent taste and texture, and have a flower and fruit aroma without additives and sucrose, and a rich milk aroma.

[0010] As a preferred technical solution of the present invention, the thermophilic Streptococcus CICC The ratio of the number of live bacteria of Lactobacillus 6063 and Lactobacillus helveticus CICC6064 is 10:1. The fermented milk prepared by using the composite starter has the highest number of live bacteria and a more obvious weak after-acid effect. The content of aldehyde compounds is as high as 19256 μg / kg, which is 3.46 times that of commercial starter. The content of ethyl ester compounds is 1641.59 μg / kg, which gives the fermented milk a richer floral and fruity aroma. In particular, the content of ethyl acetate is as high as 1559.43 μg / kg, which combines with diacetyl to bring a pleasant creamy sweetness to the fermented milk. The ratio of acetaldehyde to diacetyl (2,3-butanedione) is about 1.27:1, which can produce a typical yogurt flavor. In particular, it is found that the prepared fermented milk also contains citronellol, with a content of 150.86 μg / kg. Citronellol has a rose aroma with a threshold of 40 μg / kg, which makes the fermented milk have excellent taste and texture, and the floral and fruity aroma and milk aroma are more intense while having a rose aroma, and the sensory performance is further improved.

[0011] In a second aspect, the present invention provides a method for preparing fermented milk, the method comprising inoculating the composite starter described in the first aspect into raw milk for fermentation to obtain the fermented milk.

[0012] The preparation method provided by the present invention is simple and clean. The fermented milk prepared without adding sucrose and additives has good taste, floral and fruity aroma, rich milk flavor, fine texture, soft taste, no peculiar smell or foreign body feeling, is suitable for mass production, and has broad market prospects.

[0013] Before inoculation, the invention firstly restores the freeze-dried bacterial powders of thermophilic streptococcus CICC 6063 and lactobacillus helveticus CICC 6064 to room temperature respectively and then performs inoculation.

[0014] As a preferred technical solution of the present invention, the ratio of the number of live bacteria of Streptococcus thermophilus CICC 6063 and Lactobacillus helveticus CICC 6064 in the composite leavening agent is 1-100:1, preferably 5-20:1, preferably 5-10:1, and more preferably 10:1.

[0015] As a preferred technical solution of the present invention, the inoculation amount of thermophilic Streptococcus CICC 6063 in the composite fermentation agent is 1×10 6 -1×l0 7 CFU / mL, for example 2×10 6 ,4×l0 6 ,6×l0 6 ,8×l0 6 wait.

[0016] As a preferred technical solution of the present invention, the inoculation amount of Lactobacillus helveticus CICC 6064 in the composite starter is 1×10 5 -1×l0 6 CFU / mL, for example 2×10 5 ,4×l0 5 ,6×l0 5 ,8×l0 5 wait.

[0017] As a preferred technical solution of the present invention, the raw milk needs to be homogenized and sterilized before fermentation. The preparation method of the present invention sequentially homogenizes and sterilizes the raw milk before fermentation, so as to avoid the influence of impurities, bacteria, etc. in the system, which is more conducive to fermentation.

[0018] As a preferred technical solution of the present invention, the homogenization method is to homogenize the raw milk under the conditions of 58-62°C and 15-25MPa, wherein the 58-62°C can be 59°C, 60°C, or 61°C; the 15-25MPa can be 16MPa, 18MPa, 20MPa, 22MPa, 24MPa, etc.

[0019] As a preferred technical solution of the present invention, the sterilization method is to sterilize the raw milk at 94-96°C for 8-12 minutes, wherein the 94-96°C can be 94°C, 95°C, 96°C, etc., and the 8-12 minutes can be 9 minutes, 10 minutes, 11 minutes, etc.

[0020] As a preferred technical solution of the present invention, the raw milk includes cow's milk, goat's milk or camel's milk, preferably cow's milk.

[0021] As a preferred technical solution of the present invention, the fermentation temperature is 42-44°C, such as 42°C, 43°C, 44°C, etc., preferably 42°C.

[0022] As a preferred technical solution of the present invention, the fermentation time is 4-6 hours, such as 4.5 hours, 5 hours, 6 hours, etc., preferably 4-5 hours.

[0023] The composite fermentation agent provided by the present invention has a fast fermentation speed and can significantly shorten the fermentation time, reaching the fermentation end point in 4.2 hours at the fastest.

[0024] As a preferred technical solution of the present invention, the preparation method further comprises the step of fermenting the fermented milk to a pH value of 4.5-4.6, completing the fermentation, and storing the fermented milk at a temperature of 2-8°C.

[0025] As a preferred technical solution of the present invention, the preparation method comprises:

[0026] (1) Restoring freeze-dried bacterial powders of Streptococcus thermophilus CICC 6063 and Lactobacillus helveticus CICC 6064 to room temperature respectively;

[0027] (2) homogenizing the raw milk at 58-62°C and 15-25 MPa, and then sterilizing at 94-96°C for 8-12 min;

[0028] (3) inoculating the raw milk with a ratio of 5-20:1 of the number of live bacteria of Streptococcus thermophilus CICC 6063 and Lactobacillus helveticus CICC 6064 as a composite starter for fermentation at a temperature of 42-44° C. for 4-5 h;

[0029] (4) When the pH value of the fermented milk reaches 4.5-4.6, the fermentation is complete and the milk is stored at 2-8°C.

[0030] In a third aspect, the present invention provides a fermented milk, which is obtained by fermenting with the composite starter described in the first aspect, or prepared by the preparation method described in the second aspect.

[0031] The fermented milk prepared by the present invention is of coagulated or stirred type, has unique flavor, has floral and fruity aroma and rich milk flavor, has fine texture, soft taste, and has no peculiar smell or foreign body sensation. Compared with the fermented milk prepared by commercial starter, the sensory and fermentation characteristic indicators are similar to or even better.

[0032] As a preferred technical solution of the present invention, the fermented milk does not contain additives and sucrose. The additives are commonly used additives in the art, such as coagulants, stabilizers, thickeners and sweeteners. The fermented milk prepared without adding additives has a good taste, floral and fruity aroma and rich milk flavor, and is particularly suitable for the preparation of additive-free fermented milk.

[0033] The fermented milk provided by the invention has good palatability without adding sucrose, and is particularly suitable for the preparation of sugar-free fermented milk.

[0034] As a preferred technical solution of the present invention, the content of aldehyde compounds in the fermented milk is 19200-20500 μg / kg.

[0035] As a preferred technical solution of the present invention, the content of ethyl ester compounds in the fermented milk is 1250-1650 μg / kg.

[0036] The fermented milk prepared by the invention has a high content of ethyl ester compounds, which gives the fermented milk a floral and fruity aroma and a rich milky aroma.

[0037] As a preferred technical solution of the present invention, the mass ratio of acetaldehyde to 2,3-butanedione in the fermented milk is 1.09-1.27:1.

[0038] The mass ratio of acetaldehyde to 2,3-butanedione in the fermented milk prepared by the invention is 1.09-1.27:1, and the fermented milk has a typical yogurt flavor.

[0039] As a preferred technical solution of the present invention, the content of citronellol in the fermented milk is 100-200 μg / kg, for example, 120 μg / kg, 140 μg / kg, 160 μg / kg, 180 μg / kg, etc.

[0040] The fermented milk prepared by the present invention has citronellol without adding additives such as sweeteners, thereby enhancing the rose aroma and having rich floral and fruity aroma and milky flavor.

[0041] The technical solution provided by the embodiment of the present invention has the following advantages compared with the prior art:

[0042] 1. The strains of the composite starter provided by the present invention have a synergistic symbiotic effect. By selecting and compounding the strains, the fermentation agent has the characteristics of high viable count and post-acid stability, which helps to improve product quality, extend shelf life, and improve the flavor of fermented milk. The prepared fermented milk has a rich milky and floral and fruity aroma, a fine texture, a soft taste, and no peculiar smell or foreign body sensation.

[0043] 2. The fermentation agent provided by the present invention is used for fermentation, and the fermentation speed is fast, which can significantly shorten the fermentation time.

[0044] 3. In the present invention, when the ratio of the number of live bacteria of Streptococcus thermophilus CICC 6063 and Lactobacillus helveticus CICC 6064 is 10:1 without adding sucrose and additives, the content of aldehyde compounds in the prepared fermented milk is 19256 μg / kg, which is 3.46 times that of the commercial starter, and the ratio of acetaldehyde to diacetyl (2,3-butanedione) is about 1.27:1, which can produce a typical yogurt flavor. The citronellol content is 150.86 μg / kg, giving the fermented milk product a rich milky aroma and rose fruit aroma, a soft taste, and no peculiar smell or foreign body sensation. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the invention and, together with the description, serve to explain the principles of the invention.

[0046] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative labor.

[0047] Figure 1 This is a scanning electron microscope image of Streptococcus thermophilus CICC 6063 of Experimental Example 1 of the present invention;

[0048] Figure 2 The phylogenetic tree constructed for the thermophilic Streptococcus CICC 6063 strain in Experimental Example 1 of the present invention;

[0049] Figure 3 This is a scanning electron microscope image of Lactobacillus helveticus CICC 6064 of Experimental Example 2 of the present invention;

[0050] Figure 4 The phylogenetic tree constructed for the Lactobacillus helveticus CICC 6064 strain in Experimental Example 2 of the present invention;

[0051] Figure 5 This is a peak diagram of flavor substances detected in fermented milk prepared in Example 1 of the present invention;

[0052] Figure 6 This is a peak diagram of flavor substances detected in fermented milk prepared in Example 2 of the present invention;

[0053] Figure 7 This is a peak diagram of flavor substances detected in fermented milk prepared in Comparative Example 1 of the present invention;

[0054] Figure 8 This is a peak diagram of flavor substances detected in fermented milk prepared in Comparative Example 2 of the present invention;

[0055] Fig. 9 This is a peak diagram of flavor substances detected in fermented milk prepared in Comparative Example 3 of the present invention;

[0056] Fig.10 This is a peak diagram of flavor substances detected in fermented milk prepared in Comparative Example 4 of the present invention;

[0057] Fig.11 This is the peak diagram for detecting flavor substances in the fermented milk prepared in Comparative Example 5 of the present invention. DETAILED DESCRIPTION

[0058] In order to more clearly understand the above-mentioned objectives, features and advantages of the present invention, the scheme of the present invention will be further described below. It should be noted that the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

[0059] In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention may also be implemented in other ways different from those described herein; it is obvious that the embodiments in the specification are only part of the embodiments of the present invention, rather than all of the embodiments.

[0060] The thermophilic streptococcus CICC 6063 and the lactobacillus helveticus CICC 6064 strains of the invention are derived from the China Industrial Microbiological Culture Collection Management Center, and the collection numbers are thermophilic streptococcus CICC 6063 and lactobacillus helveticus CICC 6064.

[0061] Experimental Example 1

[0062] Identification of Streptococcus thermophilus CICC 6063.

[0063] (1) This experiment was conducted by scanning electron microscopy and physiological and biochemical tests on Streptococcus thermophilus CICC 6063. Figure 1 This is a scanning electron micrograph of Streptococcus thermophilus CICC 6063.

[0064] It can be found that the thermophilic Streptococcus CICC 6063 bacteria are spherical, with a diameter of 0.7-0.8 μm, in pairs or chains, non-spore-forming, flagella-free, non-motile, Gram-positive, and ferment lactose and sucrose, but not maltose and raffinose.

[0065] (2) Using the bacterial 16srRNA identification method, the phylogenetic tree constructed for the thermophilic Streptococcus CICC 6063 strain was as follows Figure 2 As shown by Figure 2 The strain was identified as Streptococcus thermophilus.

[0066] Experimental Example 2

[0067] (1) Identification of Lactobacillus helveticus CICC 6064

[0068] In this experiment, scanning electron microscopy and physiological and biochemical tests were performed on Lactobacillus helveticus CICC 6064. Figure 3 This is a scanning electron micrograph of Lactobacillus helveticus CICC 6064.

[0069] It can be found that Lactobacillus helveticus CICC 6064 is rod-shaped, (0.5-0.6)×(1.1-2.5)μm, spore-free, Gram-positive, and ferments glucose, lactose, and fructose, but does not ferment starch, xylose, arabinose, and sorbitol.

[0070] (2) The phylogenetic tree constructed for Lactobacillus helveticus CICC 6064 strain using the bacterial 16srRNA identification method was as follows: Figure 4 As shown by Figure 4 The strain was identified as Lactobacillus helveticus.

[0071] Example 1

[0072] This embodiment provides a composite starter, fermented milk and a preparation method thereof, comprising the following steps:

[0073] (1) Preparation of strains: The freeze-dried single-strain freeze-dried bacterial powder was taken out of the -80°C refrigerator and restored to room temperature. The live bacterial count of the freeze-dried bacterial powder of Streptococcus thermophilus CICC 6063 was 9.8×10 8 CFU / g, the number of live bacteria of freeze-dried powder of Lactobacillus helveticus CICC 6064 is 3.4×10 8 CFU / g.

[0074] (2) Raw milk homogenization and sterilization: Using whole milk as raw material, heat the milk to 60°C and 20MPa for homogenization; then sterilize at 95°C for 10 min.

[0075] (3) Inoculation and fermentation: After sterilization, the mixture was cooled to 42°C, and the ratio of the number of live bacteria of thermophilic Streptococcus CICC 6063 and Lactobacillus helveticus CICC 6064 in the composite fermentation medium was 10:1, and inoculation was performed, and the inoculation amount of thermophilic Streptococcus CICC 6063 was 5×10 6 CFU / mL, the inoculum size of Lactobacillus helveticus CICC 6064 was 5×10 5 CFU / mL, constant temperature fermentation at 42℃.

[0076] (4) When the pH value of the fermented milk reaches 4.5-4.6, the fermentation is terminated and the milk is stored at 4°C.

[0077] Example 2

[0078] The present embodiment provides a composite starter, fermented milk and a preparation method thereof. The preparation method is the same as that in Example 1. The difference from Example 1 is that the ratio of the number of live bacteria of Streptococcus thermophilus CICC 6063 and Lactobacillus helveticus CICC 6064 in the composite starter in the present embodiment is 5:1, and the total inoculation amount is the same as that in Example 1.

[0079] Comparative Example 1

[0080] This comparative example provides a starter, fermented milk and a preparation method thereof. The preparation method is the same as that in Example 1. The difference from Example 1 is that in this comparative example, the composite starter is replaced by thermophilic Streptococcus CICC 6063, and the total inoculation amount is the same as that in Example 1.

[0081] Comparative Example 2

[0082] This comparative example provides a starter, fermented milk and a preparation method thereof. The preparation method is the same as that in Example 1. The difference from Example 1 is that in this comparative example, the composite starter is replaced by Lactobacillus helveticus CICC 6064, and the total inoculation amount is the same as that in Example 1.

[0083] Comparative Example 3

[0084] This comparative example provides a starter, fermented milk and a preparation method thereof. The preparation method is the same as that in Example 1. The difference from Example 1 is that in this comparative example, the composite starter is replaced with an imported commercial starter (purchased from IFF Company), and the inoculation amount is 5.5×10 6 CFU / mL.

[0085] Comparative Example 4

[0086] This comparative example provides a composite starter, fermented milk and a preparation method thereof. The preparation method is the same as that in Example 1. The difference from Example 1 is that the ratio of the number of live bacteria of Streptococcus thermophilus CICC 6063 and Lactobacillus helveticus CICC 6064 in the composite starter in this comparative example is 1:1, and the total inoculation amount is the same as that in Example 1.

[0087] Comparative Example 5

[0088] This comparative example provides a composite starter, fermented milk and a preparation method thereof. The preparation method is the same as that in Example 1. The difference from Example 1 is that the ratio of the number of live bacteria of Streptococcus thermophilus CICC 6063 and Lactobacillus helveticus CICC 6064 in the composite starter in this comparative example is 100:1, and the total inoculation amount is the same as that in Example 1.

[0089] Comparative Example 6

[0090] This comparative example provides a composite starter, fermented milk and a preparation method thereof. The preparation method is the same as that in Example 1. The difference from Example 1 is that the ratio of the number of live bacteria of Streptococcus thermophilus CICC 6063 and Lactobacillus helveticus CICC 6064 in the composite starter in this comparative example is 1000:1, and the total inoculation amount is the same as that in Example 1.

[0091] Performance Test 1

[0092] The pH value of Example 1-2 and Comparative Example 1-6 during the fermentation process was continuously monitored using an iCinac fermentation monitor, and the pH value changes of the fermented milk obtained in Example 1-2 and Comparative Example 1-6 after storage at 4°C for 1d, 7d, 14d, and 21d were measured using a pH meter.

[0093] The results are shown in Table 1:

[0094] Table 1

[0095]

[0096] Here, 0d refers to the time point when the pH value reaches the isoelectric point of casein and is transferred to 4°C for storage.

[0097] As shown in Table 1, fermentation is completed within 4-5h using the composite fermentation agent provided by the present invention to prepare fermented milk, and the fermentation speed is the fastest at 4.2h when the ratio of the number of viable bacteria of thermophilic streptococcus CICC 6063 and lactobacillus helveticus CICC 6064 is 5-10:1; As shown in the comparison of Example 1 and Comparative Examples 1-2, the composite fermentation agent provided by the present invention can effectively shorten the fermentation time and improve the fermentation rate relative to a single strain. As shown in the comparison of Example 1 and Comparative Examples 4-5, when the ratio of the number of viable bacteria of thermophilic streptococcus CICC 6063 and lactobacillus helveticus CICC 6064 in the composite fermentation agent is within the scope of the present invention, the fermentation time can be effectively shortened and the fermentation rate can be improved. As shown in the comparison of Example 1 and Comparative Example 3, during the low temperature storage period, the pH value of the fermented milk prepared by the present invention can be maintained between 4.2-4.5, with obvious weak post-acid effect, and the fermented milk prepared by the imported commercial fermentation agent is equivalent to or even better, and the product shelf life can be extended.

[0098] Performance Test 2

[0099] Detection of the number of viable bacteria in the fermented milk prepared in Examples 1-2 and Comparative Examples 1-6.

[0100] According to the national standard GB4789.35 Food Microbiology Test for Lactic Acid Bacteria, take 25g of fermented milk sample, add it to 225mL of saline, shake it, and get a 10-fold dilution. Take 1mL of dilution, add 9mL of saline, and get a 100-fold dilution. Repeat the above steps, and gradually dilute to 10. 7 -10 8 Times. Take 1mL of the dilution into a culture dish, use MC agar medium to count streptococci, and MRS medium to count lactobacilli, pour the medium and shake it well. After solidification, culture the MC medium at 37℃ for 48h under anaerobic conditions, and culture the MRS medium at 37℃ for 72h under oxygen-sufficient conditions. Count streptococci and lactobacilli separately, and the sum of the two results is the total number of viable bacteria.

[0101] The results are shown in Table 2:

[0102] Table 2

[0103]

[0104] As shown in Table 2, under the condition of keeping the total number of viable bacteria inoculated unchanged, the composite starter culture of the present invention can effectively increase the number of viable bacteria in fermented milk. When the ratio of the number of viable bacteria of Streptococcus thermophilus CICC 6063 to that of Lactobacillus helveticus CICC 6064 is within the range of the present invention, the number of viable bacteria is 4.00×10 9 CFU / mL, especially when it is 10:1, the number of live bacteria in the fermented milk is the highest. From the comparison between Comparative Example 1 and Comparative Example 4, it can be found that when the ratio of the number of live bacteria of Streptococcus thermophilus CICC 6063 and Lactobacillus helveticus CICC6064 is 1:1, the number of live bacteria in the fermented milk cannot be effectively increased; and the number of live bacteria in the fermented milk prepared by the embodiment of the present invention is higher than the number of live bacteria in the fermented milk prepared by the imported commercial starter in Comparative Example 3. The fermented milk prepared by the composite starter of the present invention meets the requirements of the national standard for the number of live bacteria in fermented milk products (the number of live bacteria is greater than 10 6 CFU / mL).

[0105] Performance Test 3

[0106] The content of volatile flavor substances in the fermented milk prepared in Examples 1-2 and Comparative Examples 1-5 was detected.

[0107] Headspace solid phase microextraction (HS-SPME) was used to extract and enrich the volatile odor compounds in the samples, and GC-MS was used to fully identify the volatile odor compounds. The detection parameters were as follows:

[0108] Sample pretreatment: Weigh 5g of fermented milk, add 0.5g of sodium chloride and 1μL of internal standard 2-methyl-3-heptanone with a mass concentration of 0.816mg / mL. After heating at 45℃ for 30min, insert the SPME extraction head into the headspace for 30min adsorption, inject into GC-MS, and analyze at 250℃ for 5min.

[0109] GC conditions: SH-PolarWax column (60m×0.25mm, 0.25μm) was used. The temperature was programmed to be 40°C for 5 min, then raised to 250°C at a rate of 3°C / min and maintained for 15 min. The carrier gas was helium at a flow rate of 1.0mL / min in split mode.

[0110] MS conditions: electron ionization (EI) source, electron energy 70 eV; injection port temperature 250 °C, mass acquisition range (m / z) from 35 to 400. The ion source temperature was maintained at 200 °C, and the emission current was set to 100 μA.

[0111] The test results are shown in Tables 3-7 and Figure 5-11 shown.

[0112] Figure 5 This is the peak diagram of flavor substances detected in fermented milk prepared in Example 1 of the present invention. Figure 6 This is the peak diagram of flavor substances detected in fermented milk prepared in Example 2 of the present invention. Figure 7 This is the peak diagram of flavor substances detected in the fermented milk prepared in Comparative Example 1 of the present invention. Figure 8 This is the peak diagram of flavor substances detected in fermented milk prepared in Comparative Example 2 of the present invention. Fig. 9 This is the peak diagram of flavor substances detected in fermented milk prepared in Comparative Example 3 of the present invention. Fig.10 This is the peak diagram of flavor substances detected in fermented milk prepared in Comparative Example 4 of the present invention. Fig.11 This is the peak diagram for detecting flavor substances in the fermented milk prepared in Comparative Example 5 of the present invention.

[0113] Table 3 Aldehyde compounds

[0114]

[0115] Table 4 Ketone compounds

[0116]

[0117]

[0118] Table 5 Acid compounds

[0119] Table 6 Ester compounds

[0120]

[0121] Table 7 Alcohol compounds

[0122]

[0123]

[0124] As shown in Tables 3-7, the fermented milk prepared by the present invention has a floral and fruity aroma and a rich milky aroma without additives and sucrose. A total of 66 volatile substances, including aldehydes, ketones, acids, esters, and alcohol compounds, were detected in the fermented milk prepared by the composite starter, single strain, and imported commercial starter of the present invention. The volatile substances detected in Examples 1-2 and Comparative Examples 1-5 were 51, 39, 30, 39, 45, 35, and 43, respectively.

[0125] From the comparison between Examples 1-2 and Comparative Examples 1-2, it can be seen that the contents of aldehyde compounds in the fermented milk prepared in Comparative Examples 1-2 are 4.9 μg / kg and 19.35 μg / kg, respectively, and the contents of ethyl ester compounds are 0.44 μg / kg and 1.62 μg / kg, respectively, which are extremely low and do not have floral and fruity aromas; Comparative Example 1 does not contain acetaldehyde, while the acetaldehyde content in Comparative Example 2 is too high, which will produce a "fruity taste". From the comparison between Examples 1-2 and Comparative Examples 4-5, it can be seen that the contents of aldehyde compounds in the fermented milk prepared in Comparative Examples 4 and 5 are 17851.93 μg / kg and 5012.21 μg / kg, respectively, which are lower than the embodiments of the present invention, and the ratios of acetaldehyde and diacetyl (2,3-butanedione) in the fermented milk prepared in Comparative Examples 4 and 5 are 1.39 and 0.30, respectively, and neither can produce a classic yogurt flavor.

[0126] The content of aldehyde compounds in the fermented milk obtained in Example 1-2 is above 19200 μg / kg, which is higher than the content of aldehyde compounds in Comparative Example 3; the content of ethyl ester compounds in Example 1-2 is above 1250 μg / kg, which is higher than Comparative Example 3, giving the fermented milk the aroma of flowers and fruits, especially the ethyl acetate content is above 1150 μg / kg, and also makes the fermented milk have a pleasant creamy sweetness. The ratio of acetaldehyde to diacetyl (2,3-butanedione) is between 1.09-1.27:1, which can produce a typical yogurt flavor.

[0127] In particular, n-octanal, 3-octanone, propyl butyrate, ethyl valerate, etc., which were not detected in the fermented milk prepared by the imported commercial starter, were detected in the fermented milk prepared by Example 1, and the contents of acetaldehyde, 2,3-butanedione (also known as diacetyl), and acetoin, which are common volatile flavor substances in fermented milk, were 4.11 times, 1.16 times, and 1.14 times that of Comparative Example 3, respectively. The total contents of aldehydes, ketones, acids, and alcohol compounds in the fermented milk obtained in Example 1 were 19256.63 μg / kg, 51205.57 μg / kg, 64586.53 μg / kg, and 3011.95 μg / kg, respectively, which were 3.46 times, 1.06 times, 1.16 times, and 1.63 times that of the imported commercial starter, respectively. The content of ethyl ester compounds in the fermented milk obtained in Example 1 is 1641.59 μg / kg, which gives the fermented milk a richer floral and fruity aroma, especially the ethyl acetate content is as high as 1559.43 μg / kg, which combines with diacetyl to bring a pleasant creamy sweetness to the fermented milk; the ratio of acetaldehyde to diacetyl (2,3-butanedione) is about 1.27:1, which can produce a typical yogurt flavor. At the same time, the content of the unique volatile flavor compound citronellol is 150.86 μg / kg, and the threshold value of this substance reported in the literature is 40 μg / kg, which has a rose aroma. The detected substance content is about 3 times its threshold value, giving the fermented milk a rose aroma; and the unique butyrate propyl and valerate ethyl ester of the fermented milk have contents of 53.26 μg / kg and 41.05 μg / kg, respectively, and the smell of the two compounds has a fruity aroma. Under the joint action of these volatile flavor compounds, the fermented milk prepared by the composite starter of the present invention has a unique floral and fruity aroma and a strong milky flavor.

[0128] Performance Test 4

[0129] Sensory evaluation of the fermented milks prepared in Examples 1-2 and Comparative Examples 1-6.

[0130] Referring to the industry standard "RHB104-2020 Sensory Evaluation Rules for Fermented Milk", the fermented milk was evaluated after being ripened at 4°C for 1 day. A sensory evaluation panel was formed by 10 volunteers. The panel members received two 2-hour training sessions and were required to evaluate the appearance, smell, flavor and texture of the fermented milk. During the training, four well-known domestic fermented milks were provided to the sensory evaluation panel members to help them define and reference various indicators in the fermented milk. The evaluation criteria are shown in Table 8. The score sheets of the 10 volunteers were statistically calculated and the average score was shown in Table 9.

[0131] Table 8

[0132]

[0133] Table 9

[0134]

[0135]

[0136] As shown in Table 9, compared with the single-strain starter, the two strains of the composite starter of the present invention interact with each other through a variety of complex synergistic relationships, giving excellent characteristics such as fast acid production and strong characteristic flavor. When the ratio of the number of viable bacteria of Streptococcus thermophilus CICC 6063 and Lactobacillus helveticus CICC 6064 in the composite starter is within the scope of the present invention, the score of the prepared fermented milk is above 80, especially when it is 10:1, the score is the highest, and it is superior to fermented milks of other ratios in color, taste, and smell, and the sensory characteristics are close to those of imported commercial starters.

[0137] It should be noted that, in this article, relational terms such as "first" and "second" are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, the elements defined by the sentence "comprise a ..." do not exclude the existence of other identical elements in the process, method, article or device including the elements.

[0138] The above description is only a specific embodiment of the present invention, so that those skilled in the art can understand or implement the present invention. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments described herein, but should conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A composite leavening agent, characterized in that: The composite fermentation agent comprises thermophilic streptococcus CICC 6063 and lactobacillus helveticus CICC 6064, and the ratio of the number of live bacteria of thermophilic streptococcus CICC 6063 to that of lactobacillus helveticus CICC 6064 is 10:

1.

2. A method for preparing fermented milk, characterized in that: The preparation method comprises inoculating the composite starter according to claim 1 into raw milk for fermentation to obtain the fermented milk.

3. The preparation method according to claim 2, characterized in that: The inoculation amount of thermophilic Streptococcus CICC6063 in the composite fermentation agent is 1×10 6 -1×l0 7 CFU / mL; And / or, the inoculation amount of Lactobacillus helveticus CICC 6064 in the composite starter is 1×10 5 -1×l0 6 CFU / mL.

4. The preparation method according to claim 2 or 3, characterized in that: The raw milk needs to be homogenized and sterilized before fermentation; And / or, the raw milk includes cow's milk, goat's milk or camel's milk.

5. The preparation method according to claim 4, characterized in that: The raw milk is cow's milk.

6. The preparation method according to claim 4, characterized in that: The homogenization method comprises homogenizing the raw milk at 58-62° C. and 15-25 MPa; And / or, the sterilization method is to sterilize the raw milk at a temperature of 94-96° C. for 8-12 min.

7. The preparation method according to claim 2 or 3, characterized in that: The fermentation temperature is 42-44°C; And / or, the fermentation time is 4-5h.

8. A fermented milk, characterized in that: The fermented milk is obtained by fermenting with the composite starter culture according to claim 1, or is prepared by the preparation method according to any one of claims 2-7.

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