Probiotic fermented infant cheese powder with memory improving function and preparation method thereof

Abstract

The invention discloses probiotic fermented infant cheese powder with a memory improving function and a preparation method of the probiotic fermented infant cheese powder, and belongs to the field of infant food. The method comprises the following steps: (1) detecting the bacterium concentration by adopting near infrared, shunting by adopting micro-fluidic, and then carrying out differential sterilization treatment on dairy products with different bacterium concentrations by adopting an induced electric field; (2) adding the lactobacillus rhamnosus HN001 and the lactobacillus mucus CECT 5716 into the sterilized dairy product, and carrying out combined fermentation, wherein the lactobacillus rhamnosus HN001 and the lactobacillus mucus CECT 5716 are added into the sterilized dairy product; (3) centrifuging the fermented dairy product, and uniformly spraying a liquid obtained by mixing the supernatant and Arabic gum to the surface of cheese through a high-voltage electrostatic field to construct a compact film; and (4) sequentially carrying out medium-temperature heat pump drying, infrared technology drying and grinding to obtain cheese powder. The prepared cheese powder has the function of improving the memory ability, the survival rate of edible fungi of infants in the finished product is high, and the intestinal colonization ability is high.

Description

Technical Field

[0001] This invention relates to a probiotic fermented infant cheese powder with memory-enhancing function and its preparation method, belonging to the field of infant food. Background Technology

[0002] The brain, the highest level of the human nervous system, governs activities such as language, thinking, memory, and motor skills. Infancy is the most critical period for brain development, especially the first two years after birth. During this time, the brain's structure and function undergo rapid and complex dynamic changes, representing a critical window for brain development and the formation of intelligence (including memory and cognitive function), which relies on precise and comprehensive nutritional support. Nutritional intake during this stage not only affects short-term learning and memory abilities but also lays the foundation for lifelong brain function. Simultaneously, infants' digestive systems are not yet mature, and their gut microbiota is not yet stable, making them prone to problems such as low nutrient absorption efficiency and intestinal discomfort. Recent "microbe-gut-brain axis" theory further reveals that probiotics in the gut can indirectly and powerfully regulate brain development through pathways such as adjusting serum metabolites and influencing the expression of neurotransmitters. Therefore, achieving synergistic optimization of nutritional supplementation, gut health, and cognitive development has become the core direction and cutting-edge challenge in the research and development of high-end complementary foods for infants and young children.

[0003] Among numerous infant formula ingredients, cheese, as an important source of high-quality milk protein, calcium, phosphorus, and essential amino acids, has a higher protein digestibility and absorption rate than raw milk, and its calcium-to-phosphorus ratio is well-suited to the bone and teeth development needs of infants, making it an ideal ingredient for infant complementary foods. Cheese powder, in particular, with its advantages of low moisture content (usually <5%), long shelf life, easy preparation, and convenient quantitative feeding, better meets the "convenience" and "safety" requirements of infant complementary foods, and its application demand in the infant food market has continued to grow in recent years.

[0004] However, those skilled in the art recognize that existing infant cheese products and related preparation technologies still face three major unresolved issues, severely restricting their application in high-end nutritional functional products: First, the functions are limited and disconnected from the needs of brain development: Current infant cheeses have limited functions and fail to meet the specific needs of promoting brain development and improving memory. Most current cheeses focus on supplementing basic nutrients such as protein and calcium, and the probiotics mostly only regulate intestinal function by relieving diarrhea and promoting digestion. They generally lack specific strains and scientific ratios for infant neurodevelopment and memory enhancement. Second, excessive salt is often added for flavoring and preservation: 0.5% to 1.2% salt is often added during the process to adjust flavor and preserve. High sodium content can increase the burden on infants' kidneys, which contradicts the concept of health. Third, the drying process is energy-intensive or poses safety risks: Traditional hot air drying of cheese powder has the disadvantages of high energy consumption, low efficiency, and damage to product quality (such as protein denaturation). If low-temperature freeze drying is used to avoid heat damage, there may be potential biosafety risks because the temperature is not high enough to effectively inactivate all microorganisms.

[0005] Patent CN116898001A discloses a method for making freeze-dried infant cheese, and patent CN103766503A discloses a processing method for infant cheese teething sticks. Although both patents are related to infant cheese products, their technical solutions do not involve any ingredients that enhance memory, and are improvements on basic physical forms.

[0006] Patent CN118256399A discloses a Bifidobacterium longum strain that improves memory and enhances cognition, along with its products and applications. However, the Bifidobacterium longum used in this patent is not included in the "List of Strains for Infant and Toddler Foods," thus preventing its application in the processing of infant cheese products and creating a compliance barrier. A study by Slykerman, RF, aimed to investigate the effects of early probiotic supplementation with Lactobacillus rhamnosus HN001 and Bifidobacterium animalis subsp. lactis strain HN019 on children's cognition, behavior, and mood. The results showed that neither strain had any effect on children's neurocognition (Effect of early probiotic supplementation on childhood cognition, behavior, and mood arandomised, placebo-controlled trial).

[0007] Patent CN101869151A discloses an infant formula containing lactic acid and its preparation method. However, its improvement of infants' memory is mainly achieved through essential fatty acids such as docosahexaenoic acid and arachidonic acid, as well as nutrients such as lecithin, taurine, and choline.

[0008] Patent CN119488117A discloses an infant nutritional yogurt and its preparation process. It uses Bifidobacterium animalis subsp. lactis HN019, Lactobacillus fermentum CECT 5716, Lactobacillus rhamnosus HN001, and Bifidobacterium breve M-16V to prepare pasteurized raw milk fermented yogurt. It is mainly intended to overcome the problems of low nutritional value and low stirring efficiency of fresh fruit yogurt raw materials. However, the strains are inactivated after fermentation, and the efficacy of the yogurt on infant cognition and memory is not disclosed.

[0009] In summary, there is no existing cheese powder specifically designed to enhance the memory of infants and young children. There is an urgent need to develop a probiotic fermented cheese powder that meets the needs of infant brain development, conforms to their physiological characteristics, and enhances memory function. It also needs to meet the requirements of low sodium or even no salt addition, balance nutritional quality and biosafety, and ensure that all ingredients and strains strictly comply with relevant regulations and standards for infant food. Summary of the Invention

[0010] To address existing problems, this invention provides a probiotic-fermented infant cheese powder with memory-enhancing properties and its preparation method. This invention employs microfluidic coupled pulsed electric field sterilization, effectively reducing microbial levels without damaging the nutritional components of the dairy product. It uses two infant-edible bacterial strains for co-fermentation, and utilizes a fibrous whey protein-gum arabic electrostatic spray film to enhance the survival rate of the strains during maturation. Finally, it employs a medium-temperature heat pump and infrared technology for step-drying to produce the cheese powder.

[0011] The first objective of this invention is to provide a method for preparing cheese powder that improves the memory of infants and young children. The method involves co-fermenting dairy products with Lactobacillus rhamnosus HN001 and Lactobacillus fermentum CECT 5716 to obtain cheese powder.

[0012] In one embodiment of the present invention, the method includes the following steps: (1) Sterilize dairy products by using microfluidic coupled pulsed electric field to obtain sterilized dairy products; wherein, the microfluidic coupled pulsed electric field is obtained by detecting the bacterial concentration in the dairy products by a near-infrared light spectral sensor, using microfluidics to separate the high bacterial concentration area and the low bacterial concentration area, and applying different pulsed electric fields to the high bacterial concentration area and the low bacterial concentration area for sterilization. (2) Inoculate Lactobacillus rhamnosus HN001 and Lactobacillus fermentum CECT5716 into the sterilized milk obtained in step (1), and ferment to obtain fermented milk; (3) The fermented milk obtained in step (2) is centrifuged, and the precipitate obtained is cheese; the supernatant is collected, the supernatant is heated and then mixed with gum arabic to obtain a mixture, and the mixture is sprayed onto the surface of the cheese by electrostatic spraying to form a dense film, and then the cheese is aged. (4) The cheese that has matured in step (3) is first dried by medium-temperature heat pump, then dried by infrared, and ground to obtain cheese powder.

[0013] In one embodiment of the present invention, in step (1), the dairy product may be one or more of raw cow's milk, sheep's milk or camel's milk, with a fat content of 3.2 to 5.4 wt% and a protein content of 2.8 to 3.8 wt%.

[0014] In one embodiment of the present invention, in step (1), the dairy product is first dispersed into particles with a particle size of 40-80 μm by high-pressure homogenization, and the pressure is set to 20-30 MPa.

[0015] In one embodiment of the present invention, in step (1), the diameter of the microfluidic channel is 50~100 μm; with 10 2 The CFU / mL bacterial concentration is used as a threshold to identify high-concentration and low-concentration subfluids; among which... The pulsed electric field strength applied to the high bacterial concentration sub-stream was 25~30 T, the pulse frequency was 3~5 Hz, and the treatment time was 30~40 s; the pulsed electric field strength applied to the low bacterial concentration sub-stream was 15~20 T, the pulse frequency was 1~2 Hz, and the treatment time was 10~20 s.

[0016] In one embodiment of the present invention, in step (2), the total amount of Lactobacillus rhamnosus HN001 and Lactobacillus fermentum CECT 5716 added is 1~5wt% of the sterilized milk quality, and the mass ratio of Lactobacillus rhamnosus HN001 to Lactobacillus fermentum CECT 5716 is (1~10):1.

[0017] In one embodiment of the present invention, in step (2), *Lactobacillus rhamnosus* HN001 and *Lactobacillus fermentum* CECT 5716 are cultured to a bacterial concentration of 10. 9 ~10 10 CFU / L; the inoculation amount in dairy products is 10 CFU / L each. 5 ~10 6 CFU / L and 10 4 ~10 5 CFU / L.

[0018] In one embodiment of the present invention, in step (2), the fermentation conditions are: fermentation at 35~40℃ for 40~48 h until the pH drops to 3.0~4.0.

[0019] In one embodiment of the present invention, in step (2), the fermentation process is carried out at a low speed of 20 to 60 rpm, and the fermentation is terminated by cooling to below 3 to 6°C at a rate of 2 to 5°C / min after the fermentation is completed.

[0020] In one embodiment of the present invention, in step (3), the centrifugation conditions are: centrifugation at 3500~5000 rpm for 5~8 min.

[0021] In one embodiment of the present invention, in step (3), the conditions for the heat treatment are: heating at 50~90℃ for 1~2 h; the amount of gum arabic added is 3~5 wt% of the supernatant mass.

[0022] In one embodiment of the present invention, in step (3), the conditions for electrostatic spraying are: voltage of 9~20kV, flow rate of 3~6 mL / h, and spraying distance of 20~30 cm.

[0023] In one embodiment of the present invention, in step (3), the thickness of the dense film is 40~70 μm.

[0024] In one embodiment of the present invention, in step (3), the maturation conditions are: maturation for 7 to 30 days at a temperature of 18 to 22°C and a humidity of 50 to 60%.

[0025] In one embodiment of the present invention, in step (4), the conditions for drying by the medium-temperature heat pump are: drying at 40~60℃ for 2~3 h until the moisture content is 15~25 wt%.

[0026] In one embodiment of the present invention, in step (4), the conditions for infrared drying are: using infrared radiation with a wavelength of 10~25 μm, drying at 50~60℃ for 2~3 h, until the moisture content is 4~7 wt%.

[0027] In one embodiment of the present invention, in step (4), the particle size of the cheese powder obtained after grinding is 40-80 mesh and the moisture content is less than 7 wt%.

[0028] A second objective of this invention is to obtain a cheese powder prepared by the method that helps enhance memory and cognitive function.

[0029] In one embodiment of the present invention, the cheese powder contains active *Lactobacillus rhamnosus* HN001 and *Lactobacillus fermentum* CECT 5716, with a viable count of not less than 1 × 10⁻⁶. 5 The sodium content of the cheese powder is less than 0.1 wt%, and the CFU / g is less than 0.1 wt%.

[0030] A third object of the present invention is to provide a nutritional supplement comprising the cheese powder.

[0031] A fourth object of the present invention is to provide a food product comprising the nutritional supplement or the cheese powder.

[0032] In one embodiment of the present invention, the food is infant formula.

[0033] A fifth objective of this invention is to provide the use of the cheese powder or the nutritional supplement in the preparation of products that enhance memory.

[0034] In one embodiment of the present invention, the product includes pharmaceuticals and food.

[0035] In one embodiment of the present invention, the food includes infant formula and fruit puree.

[0036] Beneficial effects (1) Microfluidic coupled pulse sterilization has the advantages of being more precise, safer, better nutrient retention, lower energy consumption, and wider adaptability. It can strengthen sterilization in high-bacterial areas, avoiding the risk of insufficient local sterilization in traditional processes; it can reduce pulse energy in low-bacterial areas of dairy products, thereby reducing protein denaturation rate and retaining more active ingredients such as vitamins and immunoglobulins, making it more suitable for subsequent production of infant formula or high-end dairy products; it does not require high-energy pulses for all raw milk, thus reducing overall energy consumption; it can dynamically adjust the diversion and sterilization parameters according to the differences in raw milk materials, avoiding the instability of sterilization effect caused by fluctuations in raw materials.

[0037] (2) Lactobacillus rhamnosus HN001 and Lactobacillus fermentum CECT 5716 have good intestinal colonization ability and can significantly improve the memory ability of rats. Taking cheese co-fermented by Lactobacillus rhamnosus HN001 and Lactobacillus fermentum CECT 5716 of the present invention can significantly improve the exploration desire of rats for new objects, improve spatial memory ability and non-hippocampus dependent memory.

[0038] (3) The lactic acid produced during fermentation lowers the pH. The whey protein in the supernatant will undergo a fibrosis reaction when heated under acidic conditions. After being combined with gum arabic, it will be electrostatically sprayed to form a dense film on the surface of the cheese, which will isolate oxygen and create an anaerobic or low-oxygen environment, promoting the anaerobic maturation process of probiotics in the cheese.

[0039] (4) Medium-temperature heat pump combined with infrared cascade drying technology can improve drying efficiency. Medium-temperature heat pump drying technology will quickly reduce the weakly bound water in the material. Bound water and free water tend to increase, so the drying efficiency will decrease after a certain drying time. Infrared drying technology uses infrared rays emitted by radiation elements to transmit to the center of the material without a medium, causing internal molecular friction to generate heat, making the temperature gradient and humidity gradient consistent, improving the drying efficiency at the end of the drying process, and also reducing tissue shrinkage and damage.

[0040] (5) Both *Lactobacillus rhamnosus* HN001 and *Lactobacillus fermentum* CECT 5716 are listed in the "List of Strains that Can Be Used in Infant and Toddler Foods". Cheese powder prepared using *Lactobacillus rhamnosus* HN001 and *Lactobacillus fermentum* CECT 5716 can be used as a nutritional supplement or complementary food for infants and toddlers to improve their memory. At the same time, it can also be added as a nutritional supplement or active ingredient to foods for children, adolescents, and the elderly to help improve their memory. Attached Figure Description

[0041] Figure 1 The bacterial count (a), soluble solids content (b), fat mass fraction (c), and lactoferrin content (d) of milk after treatment with an induction electric field or pasteurization are given. Figure 2 The following parameters were used to measure the novelty exploration time (a), discrimination index (b), cognitive index (c), and conditioned fear rigidity rate (d) of rats after dietary intervention with cheese powder. Figure 3 It is a major upregulated substance in the serum metabolome; Figure 4 The content of bacterial flora in feces; Figure 5 The effect of aging process on the survival rate of bacterial strains in cheese; Figure 6 To investigate the effects of different heating and drying methods on the survival rate of bacterial strains in cheese powder. Detailed Implementation

[0042] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0043] The methods involved in the following embodiments: 1. Fermentation preparation of strains Lactobacillus rhamnosus HN001: Lactobacillus rhamnosus HN001 was inoculated into MRS liquid medium and anaerobically activated at 37°C for 48 h. The activated solution was then inoculated into MRS liquid medium at a rate of 1% (v / v) and anaerobically cultured at 37°C for 48 h, yielding a bacterial concentration of 6 × 10⁻⁶.8 ~10 9 CFU / mL bacterial suspension.

[0044] Fermentation of *Lactobacillus mucinus* CECT 5716: *Lactobacillus mucinus* CECT 5716 was inoculated into MRS liquid medium and anaerobically activated at 37°C for 48 h. The activated solution was then inoculated into MRS liquid medium at a rate of 1% (v / v) and anaerobically cultured at 37°C for 48 h, yielding a bacterial concentration of 6 × 10⁻⁶. 8 ~10 9 CFU / g bacterial solution.

[0045] 2. Microbial testing: Microbial counting was performed according to the plating method in GB 4789.2—2022 "National Food Safety Standard - Microbiological Examination of Food - Determination of Total Colony Count". Samples were diluted with 0.1% (v / v) sterile peptone solution, and the total colony count was determined using plate counting agar.

[0046] 3. Determination of soluble solids content: The soluble solids content of the sample was determined using a saccharimeter.

[0047] 4. Fat content determination: According to GB 5009.6—2016 "National Food Safety Standard - Determination of Fat in Food", the fat content of milk was determined by alkaline hydrolysis method. 5. Determination of lactoferrin content: Lactoferrin content was determined using a bovine lactoferrin ELISA kit.

[0048] 6. The colonization content in the intestine was detected using qPCR: Rat feces were collected once before and once after the start of the behavioral test (week 11 and week 13). The contents of Lactobacillus rhamnosus and Lactobacillus fermentum in the feces were detected by real-time quantitative PCR.

[0049] DNA was extracted from rat feces using the TaKaRa MiniBEST DNA kit. The total bacterial count in rat feces was detected using TB Green PremixEx Taq II FAST qPCR (Takara) fluorescent dye. Primers were purchased from Suzhou Genewiz Biotechnology Co., Ltd., and the primer sequences used are shown in Table 1.

[0050] Table 1 Primer sequences for real-time PCR

[0051] 7. The effect of testing on the levels of metabolites in serum. Blood samples were incubated at 4°C for 3 hours, then centrifuged at 3000 rpm for 15 minutes to collect serum. An 80% methanol aqueous solution containing 0.1% (v / v) formic acid was added. After vortexing, the sample was incubated on ice for 5 minutes, then centrifuged at 15000 g at 4°C for 10 minutes. A certain amount of the supernatant was collected and diluted with mass spectrometry-grade water to a methanol content of 53%. The supernatant was then centrifuged at 15000 g at 4°C for 10 minutes and collected. The supernatant was analyzed by LC-MS. Fold Change and VIP were used to screen for differential metabolites, with thresholds set as VIP > 1.0, FC > 1.2, or FC < 0.833 and P-value < 0.05.

[0052] The raw materials used in the following examples: Lactobacillus rhamnosus ( Lactobacillus rhamnosus HN001: Purchased from Shanghai Bestfood Food Development Co., Ltd.

[0053] Fermented Lactobacillus mucinus ( Limosilactobacillus fermentum CECT 5716: Purchased from Shanghai Bestfood Food Development Co., Ltd.

[0054] MRS broth medium (MRS liquid medium): purchased from Qingdao Haibo Biotechnology Co., Ltd. Weigh 52.4 g of powder, heat to dissolve in 1 L of distilled water, autoclave at 118℃ for 15 min, and set aside.

[0055] MRS culture medium (MRS plates): purchased from Qingdao Haibo Biotechnology Co., Ltd. Weigh 66.2 g of powder, heat to dissolve in 1 L of distilled water, autoclave at 121℃ for 15 min, and prepare plates by dispensing.

[0056] Rennet: with an enzyme activity of 2000 U / g, purchased from Jiangsu Libin Biotechnology Co., Ltd., and prepared using 10 mM PBS solvent.

[0057] Example 1: Preparation of Infant Cheese Powder A method for preparing a probiotic-fermented infant cheese powder with memory-enhancing properties includes the following steps: (1) 5 L of raw milk was pumped into the microfluidic processing chamber, and the milk was dispersed into more uniform small particles (40~60 μm) by high-pressure homogenization at 20 MPa. The specific absorption peaks of microorganisms in the milk were detected by a near-infrared spectroscopy sensor, and the milk was split using microfluidics. 2 CFU / mL was used as a threshold to identify high- and low-bacterial-concentration sub-streams in dairy products; the pulse intensity for high-bacterial-concentration sub-streams was 25 T, the pulse frequency was 3 Hz, and the processing time was 30 s; the pulse intensity for low-bacterial-concentration sub-streams was 15 T, the pulse frequency was 1 Hz, and the processing time was 20 s. (2) Add 5×10 to the 5 L of sterilized dairy product in step (1) at the same time. 5 CFU / L Lactobacillus rhamnosus HN001 and 5×10 4 Co-fermentation with CFU / L fermented Lactobacillus mucinus CECT 5716 was carried out at 37℃ for 48 h. (3) Centrifuge the fermented dairy product in step (2) (5000 rpm, 5 min) and collect the precipitate to obtain cheese; collect 50 mL of supernatant, heat at 60℃ for 1 h, mix with 1.5 g of gum arabic, spray evenly on the surface of cheese through a 15 kV electrostatic field, with a shell flow rate of 3 mL / h, and form a dense film with a thickness of 50 μm. Then, age for 7 days in an environment with 20℃ and 50% humidity. (4) The cheese after maturation in step (3) is first dried at 50°C with a medium-temperature heat pump for 3 h, and the moisture content of the cheese is reduced to 18 wt%. Then, it is treated with infrared drying at 50°C (wavelength 10 μm) for 3 h, and the moisture content of the cheese is reduced to 5 wt%. After grinding, cheese powder is obtained.

[0058] Example 2: Preparation of Infant Cheese Powder Same as Example 1, except that in step (1), 5 L of raw sheep milk is used as raw material, the high bacterial concentration sub-current pulse intensity is 30 T, the pulse frequency is 5 Hz, and the processing time is 40 s; the low bacterial concentration sub-current pulse intensity is 20 T, the pulse frequency is 3 Hz, and the processing time is 10 s.

[0059] Example 3: Preparation of Infant Cheese Powder Same as Example 1, except that in step (2), 5×10 6 CFU / L Lactobacillus rhamnosus HN001 and 5×10 5 CFU / L fermenting Lactobacillus mucinus CECT 5716 was used to co-ferment dairy products at 37°C for 40 h.

[0060] Example 4: Preparation of Infant Cheese Powder Same as Example 1, except that in step (3), 30 mL of supernatant was heated at 50°C for 2 h and then mixed with 1.5 g of gum arabic. The mixture was sprayed onto the surface of the cheese through a 20 kV electrostatic field to form a film. The cheese was then aged for 10 days at 20°C and 50% humidity.

[0061] Example 5: Preparation of Infant Cheese Powder Same as Example 1, except that in step (4), the precipitate is first dried at 60°C using a medium-temperature heat pump for 2 hours, and then dried at 60°C using infrared technology for 2 hours, and then ground to obtain cheese powder.

[0062] Comparative Example 1 Same as Example 1, except that the sterilization treatment in step (1) is performed by pasteurization, in order to explore the sterilization effect of microfluidic coupled pulsed electric field sterilization on dairy products: (1) Pump 5 L of raw milk into the processing chamber for pasteurization at 64 ℃ for 98 s; (2) Add 5×10 to the 5 L of sterilized dairy product in step (1) at the same time. 5 CFU / L Lactobacillus rhamnosus HN001 and 5×10 4 CFU / L fermenting Lactobacillus mucinus CECT 5716 was used to co-ferment dairy products at 37℃ for 48 hours. (3) Centrifuge the fermented dairy product in step (2) (5000 rpm, 5 min) and collect the precipitate to obtain cheese; take 50 mL of supernatant and heat it at 60℃ for 1 h and mix it with 1.5 g of gum arabic. Spray the mixture onto the surface of the cheese through a 15 kV electrostatic field with a shell flow rate of 3 mL / h. After forming a film, age it for 7 days at 20℃ and 50% humidity. (4) The cheese in step (3) is first dried at 50°C using a medium-temperature heat pump for 3 hours, and then dried at 50°C using infrared technology for 3 hours. After grinding, cheese powder is obtained.

[0063] Comparative Example 2 Same as Example 1, except for the fermentation treatment in step (2), where rennet is used to prepare cheese: (1) Pump 5 L of raw milk into the processing chamber and sterilize it using an induced electric field with a frequency of 50 kHz, a voltage of 300V, and a processing time of 8 minutes. (2) Add 0.5% (w / w) rennet to 5 L of sterilized dairy products in step (1), keep warm at 37°C for 8 hours; (3) Centrifuge the fermented dairy product in step (2) (5000 rpm, 5 min) and collect the precipitate to obtain cheese; take 50 mL of supernatant and heat it at 60℃ for 1 h and mix it with 1.5 g of gum arabic. Spray the mixture onto the surface of the cheese through a 15 kV electrostatic field with a shell flow rate of 3 mL / h. After forming a film, age it for 7 days at 20℃ and 50% humidity. (4) The cheese in step (3) is first dried at 50°C by a heat pump for 3 hours, and then dried at 50°C by infrared technology for 3 hours. After grinding, cheese powder is obtained.

[0064] Comparative Example 3 Same as Example 1, except that step (3) of embedding is omitted, in order to explore how many strains will survive after aging if the cheese strains are not coated and embedded during the aging process: (1) Pump 5 L of raw milk into the processing chamber and use an induction electric field to sterilize the milk before fermentation. The frequency is 50 kHz, the voltage is 300V, and the processing time is 8 minutes. (2) Add 5×10 to the 5 L of sterilized dairy product in step (1) at the same time. 5 CFU / L Lactobacillus rhamnosus HN001 and 5×10 4 CFU / L fermenting Lactobacillus mucinus CECT 5716 was used to co-ferment dairy products at 37℃ for 48 hours. (3) Centrifuge the fermented dairy product in step (2) (5000 rpm, 5 min), collect the precipitate, and mature it for 7 days at 20°C and 50% humidity. (4) The cheese matured in step (3) is first dried at 50°C using a medium-temperature heat pump for 3 hours, and then dried at 50°C using infrared technology for 3 hours. After grinding, cheese powder is obtained.

[0065] Comparative Example 4 Same as Example 1, except for the embedding treatment in step (4), which aims to explore the effect of ordinary hot air drying on the survival rate of the strain: (1) Pump 5 L of raw milk into the processing chamber and use an induction electric field to sterilize the milk before fermentation. The frequency is 50 kHz, the voltage is 300V, and the processing time is 8 minutes. (2) Add 5×10 to the 5 L of sterilized dairy product in step (1) at the same time. 5 CFU / L Lactobacillus rhamnosus HN001 and 5×10 4 CFU / L fermenting Lactobacillus mucinus CECT 5716 was used to co-ferment dairy products at 37℃ for 48 hours. (3) Centrifuge the fermented dairy product in step (2) (5000 rpm, 5 min) and collect the precipitate to obtain cheese; take 50 mL of supernatant and heat it at 60℃ for 1 h and mix it with 1.5 g of gum arabic. Spray the mixture onto the surface of the cheese through a 15 kV electrostatic field with a shell flow rate of 3 mL / h. After forming a film, age it for 7 days at 20℃ and 50% humidity. (4) The cheese matured in step (3) is first dried in hot air at 50°C for 12 h until a dry solid is obtained, and then ground to obtain cheese powder.

[0066] Comparative Example 5 Same as Example 1, except that fermentation with Lactobacillus mucilaginosus CECT 5716 was used alone: (1) 5 L of raw milk was pumped into the microfluidic processing chamber, and the milk was dispersed into more uniform small particles by high-pressure homogenization at 20 MPa. The specific absorption peaks of microorganisms in the milk were detected by a near-infrared spectroscopy sensor, and the milk was split using microfluidics. 2 CFU / mL was used as a threshold to identify high-bacterial and low-bacterial areas in dairy products; the high-bacterial-concentration sub-current pulse intensity was 25 T, the pulse frequency was 3 Hz, and the processing time was 30 s; the low-bacterial-concentration sub-current pulse intensity was 15 T, the pulse frequency was 1 Hz, and the processing time was 20 s. (2) Add 5×10 to the 5 L of sterilized dairy product from step (1). 4 Fermentation was carried out using CFU / L Lactobacillus mucinus CECT 5716 at 37℃ for 48 h. (3) Centrifuge the fermented dairy product in step (2) (5000 rpm, 5 min) and collect the precipitate to obtain cheese; collect 50 mL of supernatant, heat at 60℃ for 1 h, mix with 1.5 g of gum arabic, and spray evenly on the surface of cheese through a 15 kV electrostatic field with a shell flow rate of 3 mL / h. After forming a film, age for 7 days at 20℃ and 50% humidity. (4) The cheese that has matured in step (3) is first dried at 50°C by a heat pump for 3 hours, and then treated by infrared drying at 50°C for 3 hours. After grinding, cheese powder is obtained.

[0067] Comparative Example 6 Same as Example 1, except that Lactobacillus helveticus R0052 was used instead of Lactobacillus rhamnosus HN001, and co-fermented with Lactobacillus fermentum CECT 5716: (1) 5 L of raw milk was pumped into the microfluidic processing chamber, and the milk was dispersed into more uniform small particles by high-pressure homogenization at 20 MPa. The specific absorption peaks of microorganisms in the milk were detected by a near-infrared spectroscopy sensor, and the milk was split using microfluidics. 2 CFU / mL was used as a threshold to identify high-bacterial and low-bacterial areas in dairy products. For high-bacterial-concentration sub-current pulse intensity of 25 T, pulse frequency of 3 Hz, and processing time of 30 s; for low-bacterial-concentration sub-current pulse intensity of 15 T, pulse frequency of 1 Hz, and processing time of 20 s. (2) Add 5×10 to the 5 L of sterilized dairy product from step (1). 5 CFU / L Lactobacillus helveticus R0052 and 5×10 4 Co-fermentation with CFU / L fermented Lactobacillus mucinus CECT 5716 was carried out at 37℃ for 48 h. (3) Centrifuge the fermented dairy product in step (2) (5000 rpm, 5 min) and collect the precipitate to obtain cheese; collect 50 mL of supernatant, heat at 60℃ for 1 h, mix with 1.5 g of gum arabic, and spray evenly on the surface of cheese through a 15 kV electrostatic field with a shell flow rate of 3 mL / h. After forming a film, age for 7 days at 20℃ and 50% humidity. (4) The cheese that has matured in step (3) is first dried at 50°C by a heat pump for 3 hours, and then treated by infrared drying at 50°C for 3 hours. After grinding, cheese powder is obtained.

[0068] Test case 1. Detection of bacterial count and physicochemical properties of sterilized dairy products in Example 1 and Comparative Example 1 The results are as follows Figure 1 As shown, pasteurization and the sterilization treatment in Example 1 had no significant effect on the soluble solids, fat content, and lactoferrin content of milk, with only slight fluctuations. However, the content of residual bacterial strains in pasteurized milk was significantly higher than that in milk sterilized in Example 1. It is foreseeable that milk sterilized in Example 1 will have a longer shelf life and be less prone to spoilage than pasteurized milk.

[0069] 2. Effects of Example 1 on individual memory ability in rats Dietary intervention experiments with cheese powder were conducted on rats in Examples 1, 2, 5, and 6, respectively. It was found that the intervention group in Example 1 had improved memory and could colonize in the intestine.

[0070] Mouse grouping: Sixty healthy male SD rats aged 6 weeks and weighing 20±1g were randomly divided into three groups (n=12): a control group, the intervention group of Example 1, and the intervention groups of Comparative Examples 2, 5, and 6. The experiment was divided into two phases: an adaptation period (1 week, free access to food and water) and a probiotic intervention period (8 weeks). All rats in each group were fed a normal diet. After 1 week of adaptation, the rats in the two intervention groups were administered 0.2 g of cheese powder (dissolved in 1 mL of sterile water, with a bacterial concentration of 10) by gavage daily. 4 Rats in the control group were administered the same volume of sterile water by gavage (CFU / g). Behavioral tests were conducted starting at week 12 after the probiotic intervention ended.

[0071] (1) Y-maze test: This test consists of two experiments, 2 hours apart. The first experiment is the acquisition phase, in which one arm is closed and the rat is allowed to explore freely in the other two arms for 5 minutes. Two hours later, the second experiment (recall phase) is conducted, in which all arms are opened and the rat is allowed to move freely in the three arms for 5 minutes. VisuTrack software is used to track the rat's movement and record the time and distance traveled in each arm. The recorded parameters include the number of times the rat enters each arm and the time and distance traveled in each arm.

[0072] (2) New object recognition experiment: The day before training, the test rats were allowed to explore the test box for 10 minutes to adapt; during training, two identical objects were placed in the same test box, and the test rats were allowed to explore the test box for 5 minutes; 24 hours later, one of the objects was replaced with a new object, and the test rats were placed in the test box for 5 minutes and the video was recorded. The video was analyzed using VisuTrack software to examine whether the rats could recognize one of the objects as a new object. The time taken for the rats to smell each object was recorded, and the cognitive index and discrimination index were evaluated. The formula is as follows: ; .

[0073] (3) Conditioned fear experiment: Establishment of conditioned fear: On day 1, rats were placed in an experimental chamber (with a copper grid at the bottom that could be electrified). After acclimatization for 3 minutes, a single-frequency sound stimulus (3.0 kHz, 65 dB, 28 s) was given, followed simultaneously by an inescapable foot shock (0.7 mA, 2 s). The sound and shock ended simultaneously. After the experiment, the rats remained in the chamber for another 2 minutes before being returned to their cages. The bottom of the chamber was wiped with 75% alcohol after each experiment. 48 hours later, rats with established conditioned fear were placed back in the original chamber without any stimulus for 3 minutes. 2 hours later, the chamber environment was changed (a whiteboard was placed at the bottom, and the color of the chamber walls was changed), and the rats were placed back in the chamber. After acclimatization, the same intensity sound stimulus was given for 3 minutes, and the rats' cue freezing behavior (mainly reflecting non-hippocampal dependent memory) was recorded within 3 minutes.

[0074] Dietary intervention experiments with cheese powder were conducted on rats in Example 1 and Comparative Example 2, respectively. The results showed that the intervention group in Example 1 had improved memory and could colonize the intestines. (1) The Y-maze experiment shows that ( Figure 2a): Example 1 significantly increased the exploration time of rats in the novel arm by 70% compared to the control group, indicating that Example 1 can significantly enhance the spatial memory ability of rats. However, there was no significant difference in the exploration time of rats in the novel arm in the interventions of Comparative Examples 2, 5, and 6 compared to the control group, meaning that the interventions of Comparative Examples 2 (unfermented cheese), 5 (single-strain fermented cheese), and 6 (other combinations of fermented cheese) could not improve the spatial memory ability of rats.

[0075] (2) The results of the new object recognition experiment show that ( Figure 2 bc): Example 1 significantly improved the discrimination index and cognitive index of rats, by 62% and 28% respectively compared with the control group. However, the discrimination index and cognitive index of rats treated with Comparative Examples 2, 5 and 6 were not significantly different from those of the control group, that is, Comparative Examples 2, 5 and 6 could not improve the spatial memory ability of rats.

[0076] (3) The results of the conditioned fear experiment showed that ( Figure 2 d): The proportion of conditioned fear rigidity in the intervention group of Example 1 was 116% higher than that in the control group, indicating that Example 1 can significantly enhance non-hippocampal dependent memory in rats. However, there was no significant difference in the proportion of conditioned fear rigidity in rats intervened in Comparative Examples 2, 5, and 6 compared with the control group, meaning that Comparative Examples 2, 5, and 6 interventions could not improve non-hippocampal dependent memory in rats.

[0077] (4) Compared with the control group, the serum glycerophospholipids in the intervention group of Example 1 were significantly upregulated, including LPA22:6, LPS20:4 and LPS20:5, which increased by 2.6 times, 3.2 times and 1.8 times, respectively. Figure 3 (), while there were no significant changes in the intervention groups in proportions 2, 5 and 6.

[0078] (5) The determination of fecal microbiota content showed that the content of Lactobacillus rhamnosus HN001 and Lactobacillus fermentum CECT 5716 in the feces of rats in the intervention group of Example 1 was significantly higher than that before the intervention. Figure 4 This indicates that these two strains of bacteria can effectively colonize the intestines.

[0079] 3. Detect the bacterial strain content in cheese powder. The bacterial count in the aged cheese of Example 1 and Comparative Example 3, and the dried cheese powder of Example 1 and Comparative Example 4, was determined: 3 g of cheese (or 1 g of cheese powder) was added to 10 mL of PBS, stirred at 100 rpm for 5 min, appropriately diluted, and 50 μL of the liquid was added to an MRS plate. The plates were then anaerobically cultured at 37°C for 24 h, and bacterial counts were performed to calculate the bacterial survival rate. The bacterial count after fermentation in step (2) of Examples 1, 3, and 4 was taken as 100%.

[0080]

[0081] Comparative Example 3 showed that only 28.12% of the strains survived after maturation, lower than the 72.81% in Example 1. This indicates that spraying fibrous whey-ginger arabic before maturation can form a film on the cheese surface, isolating oxygen and significantly improving the survival rate of the strains during maturation. Figure 5 ).

[0082] In Comparative Example 4, only 12.97% of the strains survived, lower than the 53.16% in Example 4, indicating that the heat pump-infrared drying method helps preserve strain viability. Figure 6 ).

[0083] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A method for preparing cheese powder that improves infants' and young children's memory, characterized in that, The method involves co-fermenting dairy products with *Lactobacillus rhamnosus* HN001 and *Lactobacillus fermentum* CECT 5716 to obtain cheese powder; wherein, The method includes the following steps: (1) Sterilize dairy products by using microfluidic coupled pulsed electric field to obtain sterilized dairy products; wherein, the microfluidic coupled pulsed electric field is obtained by detecting the bacterial concentration in the dairy products by a near-infrared light spectral sensor, using microfluidics to separate the high bacterial concentration area and the low bacterial concentration area, and applying different pulsed electric fields to the high bacterial concentration area and the low bacterial concentration area for sterilization. (2) Inoculate Lactobacillus rhamnosus HN001 and Lactobacillus fermentum CECT5716 into the sterilized milk obtained in step (1), and ferment to obtain fermented milk; (3) The fermented milk obtained in step (2) is centrifuged, and the precipitate obtained is cheese; the supernatant is collected, the supernatant is heated and then mixed with gum arabic to obtain a mixture, and the mixture is sprayed onto the surface of the cheese by electrostatic spraying to form a dense film, and then the cheese is aged. (4) The cheese that has matured in step (3) is first dried by medium-temperature heat pump, then dried by infrared, and ground to obtain cheese powder.

2. The method according to claim 1, characterized in that, In step (1), the dairy product is first dispersed into particles with a diameter of 40-80 μm by high-pressure homogenization and then sterilized.

3. The method according to claim 1, characterized in that, In step (1), with 10 2 The high and low bacterial concentration areas were separated using CFU / mL bacterial concentration as the threshold. The pulsed electric field intensity applied to the high bacterial concentration area was 25-30 T, the pulse frequency was 3-5 Hz, and the treatment time was 30-40 s. The pulsed electric field intensity applied to the low bacterial concentration area was 15-20 T, the pulse frequency was 1-2 Hz, and the treatment time was 10-20 s.

4. The method according to claim 1, characterized in that, In step (2), the inoculation amounts of *Lactobacillus rhamnosus* HN001 and *Lactobacillus fermentum* CECT 5716 in the dairy product were each independently 10. 5 ~10 6 CFU / L and 10 4 ~10 5 CFU / L.

5. The method according to claim 1, characterized in that, In step (3), the heating conditions are: heating at 50~90℃ for 1~2 h; the amount of gum arabic added is 3~5 wt% of the supernatant mass; and the thickness of the dense film is 40~70 μm.

6. The method according to claim 1, characterized in that, In step (4), the conditions for medium-temperature heat pump drying are: drying at 40~60℃ for 2~3 h; the conditions for infrared drying are: using infrared radiation with a wavelength of 10~25 μm, drying at 50~60℃ for 2~3 h.

7. Cheese powder prepared by any one of claims 1 to 6.

8. A nutritional supplement, characterized in that, The nutritional supplement includes the cheese powder of claim 7.

9. A food product, characterized in that, The food product includes the nutritional supplement of claim 8 or the cheese powder of claim 7; wherein... The food in question is complementary food for infants and young children.

10. The use of the cheese powder of claim 7 or the nutritional supplement of claim 8 in the preparation of a product for improving memory; optionally, the product includes a pharmaceutical or a food.

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