Collagen peptide product capable of delaying senescence and probiotic fermentation production method thereof

Abstract

The invention belongs to the technical field of food biology, and particularly relates to a collagen peptide product capable of delaying senescence and a probiotic fermentation production method of the collagen peptide product. Aiming at the industrial generality technical problems of poor sensory quality, low biological activity and many food additives of a formula type collagen peptide product sold in the market at present, the collagen peptide is used as a raw material, blueberry juice is used as a carrier, and plant lactobacillus and kluyveromyces marxianus are used as strains; the fermentation type collagen peptide product which is rich in probiotics and anthocyanin and contains no water and no additive is created by utilizing technologies such as probiotic metabolism conversion, flavor quantitative recombination and function targeted regulation and control. The product is purplish red and bright in color, uniform and stable in texture, sour, sweet and palatable in taste, harmonious and rich in fragrance and high in sensory quality; zero additive is realized, and the food safety is high; and through'zero addition of water ', metabolism and transformation of probiotics and a multi-component synergistic effect, the anti-aging effect is high.

Description

Technical Field

[0001] The invention belongs to the field of food biotechnology, and specifically relates to a collagen peptide product for delaying aging and a probiotic fermentation production method thereof. Technical Background

[0002] Collagen peptides (CPs) have a long history of research and application in the food field and are now widely used in nutritional enhancers, dietary supplements, functional foods and special medical formula foods. There are many brands of CPs products on the market today, and they come in various forms (drinks, capsules, compressed candies, solid beverages, etc.), but they are all "formulated" foods, with common technical problems in the industry such as poor sensory quality, low biological activity, and many food additives. This has led to the industry's low technological content, lack of high-end products, insufficient innovation, and serious homogeneity. This has not only restricted the industry's sustainable and high-quality development, but has also led to a prominent contradiction between supply and demand between people's demands for flavorful and healthy foods and the market's matching product supply. Therefore, in order for CPs to truly serve human nutrition and health, there are still many problems to be solved.

[0003] In terms of technology, enzymatic digestion is the most commonly used production method for CPs, which has the characteristics of mild reaction conditions, easy process control, strong site specificity, high product safety, and easy industrialization, and meets the production needs to a certain extent. However, the CPs have poor color (light yellow or brownish yellow), strong bitterness (containing bitter amino acids and peptides), and strong fishy smell (mainly from odorous substances in the raw materials and lipid oxidation and decomposition), which is difficult to meet the sensory quality requirements of modern food, and seriously limits its application in market terminal products.

[0004] In terms of products, the CPs products currently on the market usually have the following problems: 1) They are supplemented with animal and plant extracts and nutritional enhancers (vitamins, vitamin-like substances, minerals and unsaturated fatty acids, etc.), and the interactive (synergistic or antagonistic, etc.) effects are difficult to clarify; 2) They contain a large amount of food additives (pigments, flavors, preservatives, sweeteners, thickeners, stabilizers and antagonists, etc.), which pose a food safety risk; 3) One product has multiple functions and is not highly specific, which is not conducive to the choice of ordinary consumers and the enhancement of product value.

[0005] In recent years, the health effects of blueberries and probiotics have received increasing attention and favor. Blueberries are recognized as the "Queen of Fruits" in the world, one of the five healthy fruits recommended by FAO, and the only fruit among the top ten healthy foods recommended by WHO. At the same time, Lactobacillus plantarum (LP) and Kluyveromyces marxianus (KM) are both probiotics suitable for food. They grow fast, have a wide range of fermentation substrates, have good aroma production performance, and have multiple important biological functions. They can not only give products a unique taste and flavor, but also improve product stability and shelf life. Summary of the invention

[0006] In response to the common technical problems in the industry of poor sensory quality, low biological activity and many food additives of formulated CPs products on the market today, the present invention uses CPs as raw material, blueberry juice as a carrier, LP and KM as strains, and utilizes technologies such as probiotic metabolic transformation, flavor quantitative reconstruction and functional targeted regulation to create a fermented CPs product rich in probiotics and anthocyanins, with 0 added water and 0 additives. The product has good physical and chemical characteristics, sensory quality, food safety and anti-aging effects, and has achieved substantial technical effects.

[0007] In order to achieve the above objectives, the present invention adopts the following technical solutions:

[0008] A probiotic fermentation production method for anti-aging collagen peptide products, the steps are as follows

[0009] (1) Lactobacillus plantarum (LP) and Kluyveromyces marxianus (KM) were activated and expanded, respectively, and the precipitates were collected by centrifugation after cultivation; physiological saline was added to the precipitates for washing by oscillation, and the precipitates were centrifuged again after washing. The obtained precipitates were recorded as LP bacterial sludge and KM bacterial sludge, respectively.

[0010] (2) Wash the blueberries, drain the water, and grind them to obtain blueberry pulp; then add pectinase to the blueberry pulp, stir it continuously for enzymolysis, and centrifuge it after enzymolysis to obtain the supernatant, which is the blueberry juice.

[0011] (3) adding collagen peptides (CPs) to the blueberry juice prepared in step (2), stirring and dissolving to obtain a mixture of blueberry juice and CPs, and heating and sterilizing the mixture; cooling the mixture after sterilization, and then adding the LP bacterial slurry prepared in step (1) after cooling, and performing a primary fermentation under the conditions of mechanical stirring and air flow; after the primary fermentation, adding the KM bacterial slurry prepared in step (1), and performing a secondary fermentation under the conditions of mechanical stirring and air flow; and after the secondary fermentation, post-ripening, sterilization, and filling are performed to obtain a CPs product that delays aging.

[0012] Preferably, the culture medium used for activation and expansion culture of Lactobacillus plantarum in step (1) is MRS culture medium, the inoculation amount is 1-3%, the culture temperature is 30-37° C.; the activation culture time is 16-20 hours, and the expansion culture time is 8-12 hours.

[0013] Preferably, the culture medium used for activation and expansion culture of Kluyveromyces marxianus in step (1) is YM medium, the inoculation amount is 1-3%, the culture temperature is 22-28°C; the activation culture time is 12-16h, and the expansion culture time is 6-10h.

[0014] Preferably, the temperature of the physiological saline in step (1) is 4-10°C, and the amount added is 3-5L of physiological saline per kg of precipitation; the equipment used for the centrifugation is an intermittent centrifuge, the centrifugal force is 5000-8000g, and the time is 10-20min.

[0015] Preferably, the equipment used for grinding in step (2) is a colloid mill, the temperature of the colloid mill for treating blueberries is 5-15° C., and the time is 5-10 min; the equipment used for centrifugation is a tubular centrifuge, the centrifugal force is 15000-20000 g, and the time is 3-7 min.

[0016] Preferably, the amount of pectinase added in step (2) is 0.1-0.2% (w / v, g / mL) of the blueberry pulp; the enzymatic hydrolysis time is 1-2 hours, and the temperature is 45-55°C.

[0017] Preferably, the amount of CPs added in step (3) is 10-15% (w / v, g / mL) of the blueberry juice; the sterilization temperature is 70-90°C and the time is 20-30 min; and cooling refers to cooling to 30-37°C.

[0018] Preferably, the inoculation amount of the LP bacterial sludge in step (3) is 0.1-0.2 g / L based on the volume of the mixture of blueberry juice and CPs; the temperature of the primary fermentation is 30-37° C., the time is 9-12 h, the stirring speed is 120-200 rpm, and the ventilation volume is 0.2-0.3 v / v·min.

[0019] Preferably, the inoculation amount of the KM bacterial sludge in step (3) is 0.1-0.2 g / L based on the volume of the mixture of blueberry juice and CPs; the temperature of the secondary fermentation is 22-28° C., the time is 10-15 h, the stirring speed is 120-200 rpm, and the ventilation volume is 0.1-0.2 v / v·min.

[0020] Preferably, the post-ripening temperature in step (3) is 2-6°C and the time is 6-12h; the sterilization method is UHT (ultra-high temperature instantaneous sterilization), the sterilization temperature is 130-140°C, and the sterilization time is 5-10s.

[0021] The CPs described in the above technical solution are commercially available food-grade CPs, including fish-derived CPs, pig-derived CPs, cattle-derived CPs and poultry-derived CPs.

[0022] Beneficial Effects of the Invention

[0023] 1. The CPs product of the present invention has high sensory quality. Due to poor color, strong bitterness and strong fishy smell, the current CPs products on the market are generally difficult to meet consumers' demand for sensory quality. The present invention utilizes the masking effect of blueberry juice and the metabolic transformation effect of microorganisms to obtain a fermented CPs product with bright purple-red color, uniform and stable texture, sweet and sour taste, and rich and harmonious aroma, which has good sensory quality.

[0024] 2. The CPs product of the present invention has high food safety. Currently, CPs products on the market are usually supplemented with animal and plant extracts, nutritional enhancers (vitamins, minerals, unsaturated fatty acids, etc.) and food additives (pigments, flavors, preservatives, sweeteners, thickeners, stabilizers, antagonists, etc.), and long-term consumption may pose safety risks. The present invention uses ordinary food blueberry juice as a carrier and probiotics recognized by the National Health Commission as a strain to create a "0 additive" fermented CPs product with good food safety.

[0025] 3. The biological efficacy of the CPs product of the present invention is high. Due to the unsatisfactory efficacy, the anti-aging effect of CPs products has been controversial and questioned. The present invention significantly improves the biological activity of the product through the following three approaches: 1) "0" added water: using pure blueberry juice as a carrier without adding additional water, the high density of anthocyanins, vitamins, minerals, organic acids and other nutritional functional components in blueberry juice is maintained; 2) Introduction of probiotics: LP and KM not only have a direct anti-aging effect, but also generate a variety of bioactive substances through metabolic transformation, further improving the efficacy of the product; 3) Utilization of synergistic effects: Based on the synergistic effect between multiple components such as CPs, probiotics and their metabolic transformation products, and nutritional functional components of blueberry juice, an unexpected anti-aging effect is achieved. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The growth curves of probiotic activation and expansion culture (A: LP; B: KM). DETAILED DESCRIPTION

[0027] The present invention is further described in detail with reference to the following examples.

[0028] (1) Description of bacterial strains: Lactobacillus plantarum (LP) and Kluyveromyces marxianus (KM) can be obtained through commercial channels. The LP and KM used in this implementation case were purchased from Beina Chuanglian Biotechnology Co., Ltd. in the mall, with serial numbers BNCC185392 and BNCC187341, respectively.

[0029] (2) Culture medium description: Both MRS and YM culture media can be obtained through commercial channels. The MRS and YM culture media used in this example were purchased from Hangzhou Best Biotechnology Co., Ltd.

[0030] (3) Description of CPs: CPs include food-grade fish, cattle, pig and poultry CPs, all of which can be obtained through commercial channels. The CPs used in this implementation case were purchased from Shaanxi Kanglichuan Biotechnology Co., Ltd.

[0031] Note: Unless otherwise specified, the materials or reagents used in the present invention can be obtained through commercial channels; the "approximately" used in this embodiment does not mean that the meaning is unclear, but there are problems such as decimal point values, so the expression "approximately" is used, but the error is within ±0.5%.

[0032] Embodiment 1:

[0033] (1) Take 0.4 mL of LP bacterial solution frozen in glycerol and add it to 20 mL of MRS medium, and activate and culture it at 35°C for 18 hours to obtain activated bacteria; then add 1000 mL of MRS medium and expand the culture at 35°C for 10 hours; after the culture, use an intermittent centrifuge to centrifuge at 6000g for 15 minutes to obtain the precipitate, add 400 mL of 7°C physiological saline to the precipitate and shake and wash it; after washing, centrifuge at 6000g for 15 minutes to obtain the precipitate, and obtain LP bacterial sludge (about 93 g).

[0034] At the same time, 0.4 mL of KM bacterial solution frozen in glycerol was added to 20 mL of YM medium, and activated and cultured at 25°C for 14 h to obtain activated bacteria; then 1000 mL of YM medium was added, and the culture was expanded at 25°C for 8 h; after culture, the precipitate was obtained by centrifugation at 6000 g for 15 min using an intermittent centrifuge, and 400 mL of 7°C physiological saline was added to the precipitate for shaking and washing; after washing, the precipitate was obtained by centrifugation at 6000 g for 15 min, which was the KM bacterial sludge (about 98 g).

[0035] (2) 8 kg of fresh blueberries were washed and drained, and ground using a colloid mill at 10°C for 8 min to obtain blueberry pulp; then 12 g of pectinase was added to the blueberry pulp, and the pulp was continuously stirred for enzymolysis at 50°C for 1.5 h. After enzymolysis, the pulp was centrifuged at 18,000 g for 5 min using a tubular centrifuge, and the resulting supernatant was blueberry juice (about 6.2 L).

[0036] (3) adding 0.78 kg of food-grade fish-derived CPs to the blueberry juice prepared in step (2), stirring and dissolving in a 10 L fermentation tank, and then steam heating to 80° C. for sterilization for 25 min; after sterilization, cooling to 35° C., inoculating the LP sludge prepared in step (1) (inoculation amount of about 0.15 g / L), and performing a primary fermentation for 10 h at a temperature of 35° C., a stirring speed of 160 rpm, and an air ventilation volume of 0.25 v / v·min; after the primary fermentation, inoculating the KM sludge prepared in step (1) (inoculation amount of about 0.15 g / L), and performing a secondary fermentation for 12 h at a temperature of 25° C., a stirring speed of 160 rpm, and an air ventilation volume of 0.15 v / v·min; after the secondary fermentation, cooling to 4° C. with circulating water, and then ripening for 8 h, and finally UHT sterilizing at 135° C. for 7 s, and aseptically filling in a light-proof container to obtain a CPs product (about 6.6 L) with anti-aging effect.

[0037] Embodiment 2:

[0038] (1) Take 1.4 mL of LP bacterial solution frozen in glycerol and add it to 45 mL of MRS medium, activate and culture it at 30°C for 16 h to obtain activated bacteria; then add 1500 mL of MRS medium and expand the culture at 30°C for 8 h; after culture, use an intermittent centrifuge to centrifuge at 8000g for 10 min to obtain a precipitate, add 500 mL of 4°C physiological saline to the precipitate and shake and wash; after washing, centrifuge at 8000g for 10 min to obtain a precipitate to obtain LP bacterial sludge (about 141 g).

[0039] At the same time, 1.4 mL of KM bacterial solution frozen in glycerol was added to 45 mL of YM medium, and activated and cultured at 22°C for 12 h to obtain activated bacteria; then 1500 mL of YM medium was added, and the culture was expanded at 22°C for 6 h; after culture, the precipitate was obtained by centrifugation at 8000 g for 10 min in an intermittent centrifuge, and 500 mL of 4°C physiological saline was added to the precipitate for shaking and washing; after washing, the precipitate was obtained by centrifugation at 8000 g for 10 min, which was the KM bacterial sludge (about 147 g).

[0040] (2) 8 kg of fresh blueberries were washed and drained, and ground using a colloid mill at 15° C. for 5 min to obtain blueberry pulp; then 16 g of pectinase was added to the blueberry pulp, and the pulp was continuously stirred at 45° C. for 2 h for enzymolysis. After enzymolysis, the pulp was centrifuged at 20,000 g for 3 min using a tubular centrifuge, and the resulting supernatant was blueberry juice (about 6.5 L).

[0041] (3) Add 0.98 kg of food-grade bovine CPs to the blueberry juice prepared in step (2), stir and dissolve in a 10 L fermentation tank, and then steam heat to 90 ° C for sterilization for 20 min; after sterilization, cool to 30 ° C, inoculate the LP sludge prepared in step (1) (inoculation amount of about 0.2 g / L), and perform primary fermentation for 9 h at a temperature of 30 ° C, a stirring speed of 120 rpm, and an air ventilation volume of 0.2 v / v·min; after the primary fermentation, inoculate the KM sludge prepared in step (1) (inoculation amount of about 0.2 g / L), and perform secondary fermentation for 10 h at a temperature of 22 ° C, a stirring speed of 120 rpm, and an air ventilation volume of 0.1 v / v·min; after the secondary fermentation, cool to 6 ° C using circulating water and ripen for 6 h, and finally UHT sterilize at 140 ° C for 5 s, and aseptically fill in a light-proof container to obtain a CPs product (about 7.0 L) with anti-aging effect.

[0042] Embodiment 3:

[0043] (1) Take 0.1 mL of LP bacterial solution frozen in glycerol and add it to 10 mL of MRS medium, and activate and culture it at 37°C for 20 h to obtain activated bacteria; then add 700 mL of MRS medium and expand the culture at 37°C for 12 h; after culture, use an intermittent centrifuge to centrifuge at 5000g for 20 min to obtain a precipitate, add 350 mL of 10°C physiological saline to the precipitate and shake and wash; after washing, centrifuge at 5000g for 20 min to obtain a precipitate to obtain LP bacterial sludge (about 70 g).

[0044] At the same time, 0.1 mL of KM bacterial solution frozen in glycerol was added to 10 mL of YM medium, and activated and cultured at 28°C for 16 h to obtain activated bacteria; then 700 mL of YM medium was added, and the culture was expanded at 28°C for 10 h; after culture, the precipitate was obtained by centrifugation at 5000 g for 20 min using an intermittent centrifuge, and 350 mL of 10°C physiological saline was added to the precipitate for shaking and washing; after washing, the precipitate was obtained by centrifugation at 5000 g for 20 min, which was the KM bacterial sludge (about 72 g).

[0045] (2) 8 kg of thawed blueberries were washed and drained, and ground using a colloid mill at 5°C for 10 min to obtain blueberry pulp; then 8 g of pectinase was added to the blueberry pulp, and the blueberry pulp was continuously stirred and hydrolyzed at 55°C for 1 h. After the hydrolysis, the blueberry pulp was centrifuged at 15,000 g for 7 min using a tubular centrifuge. The resulting supernatant was blueberry juice (about 6.3 L).

[0046] (3) 0.63 kg of food-grade porcine CPs was added to the blueberry juice prepared in step (2), stirred and dissolved in a 10 L fermentation tank, and then steam-heated to 70 ° C for sterilization for 30 min; after sterilization, the LP sludge prepared in step (1) was added when cooled to 37 ° C, and a primary fermentation was carried out for 12 h at a temperature of 37 ° C, a stirring speed of 200 rpm, and an air ventilation volume of 0.3 v / v·min; after the primary fermentation, the KM sludge prepared in step (1) was added (agent amount of about 0.1 g / L), and a secondary fermentation was carried out for 15 h at a temperature of 28 ° C, a stirring speed of 200 rpm, and an air ventilation volume of 0.2 v / v·min; after the secondary fermentation, the mixture was cooled to 2 ° C using circulating water and then ripened for 12 h, and finally UHT sterilized at 130 ° C for 10 s, and aseptically filled in a light-proof container to obtain a CPs product (about 6.4 L) with anti-aging effect.

[0047] Performance Testing

[0048] The CPs product obtained in Example 1 was selected for subsequent performance testing.

[0049] Test method:

[0050] (1) Microbial count

[0051] Before UHT sterilization, the total bacterial count of this product is tested according to GB 4789.2-2022 "Determination of total bacterial count in food microbiological examination", and the KM count is tested according to GB 4789.15-2010 "Count of mold and yeast in food microbiological examination". The LP count is the difference between the total bacterial count and the KM count.

[0052] (2) Physical and chemical properties testing

[0053] The physical properties, chemical composition and volatile compounds of the product were tested after UHT sterilization, as follows: hydroxyproline was based on GB / T 9695.23-2008 "Determination of hydroxyproline content in meat and meat products", and the conversion coefficient between CPs and hydroxyproline was 10.0; pH was based on GB / T 10468-1989 "Determination of pH value of fruit and vegetable products"; titratable acid was based on GB12456-2021 "Determination of total acid in food"; soluble solids were based on NY / T 2637-2014 "Determination of soluble solids content in fruits and vegetables"; color was tested using a colorimeter; viscosity was tested by a rheometer at 4°C and 50·s -1 The total sugar and reducing sugar were determined by anthrone colorimetric method and dinitrosalicylic acid method respectively. The total phenols were determined by the folin phenol method and expressed as gallic acid equivalents. The total flavonoids were determined by nitrite-aluminum nitrate-sodium hydroxide colorimetric method and expressed as rutin equivalents. Anthocyanins were determined by pH differential method and expressed as cyanidin-3-glucoside equivalents.

[0054] The volatile compounds were determined by GCMS-TQ8040 HS-SPME-GC / MS produced by Shimadzu Company of Japan. The specific parameters were as follows: the sample was equilibrated at 50°C for 20 min and then headspace extracted for 30 min; the extraction head was DVB / CAR / PDMS (50 / 30 μm), and the chromatographic column was Rtx-WAX (30.0 cm×0.25 mm×0.25 μm); the initial temperature was 40°C, the injection port temperature was 250°C, and the carrier gas flow rate was 1 mL / min; the column oven temperature program was 40°C for 4 min, then increased to 160°C at 5°C / min and maintained for 4 min, then increased to 200°C at 10°C / min and maintained for 13 min, and finally increased to 240°C at 20°C / min and maintained for 5 min; the ion source temperature was 200°C, the transfer line temperature was 250°C, and semi-quantitative analysis was performed using 3-octanol as the standard.

[0055] Table 1 Quantitative description test scoring criteria for CPs products of the present invention

[0056]

[0057] (3) Sensory quality analysis

[0058] With reference to GB 7101-2022 "Sensory requirements for beverages", GB17325 "Concentrates (juices, pulps) for food industry", GB / T 31121-2014 "Fruit and vegetable juices and beverages", GB / T 32783-2016 "Blueberry wine" and DBS23 / 002-2021 "Blueberry juice beverages", 11 trained evaluators (4 males and 7 females, aged 18-47) were selected for quantitative description testing (Table 1). In addition, 30 consumer evaluators (13 males and 17 females, aged 18-52) were selected to conduct emotional (preference) testing in the laboratory using a percentage system.

[0059] (4) Food safety evaluation

[0060] Food safety evaluation was conducted with reference to GBZ / T 240.15-2011 "Chemical Toxicology Evaluation Procedures and Test Methods Part 15: Subacute Oral Toxicity Test". Twenty 6-week-old SPF female ICR mice were randomly divided into two groups (control group and treatment group); after 7 days of adaptation, the mice in the treatment group were gavaged with 20 mL / kg·bw each time, once in the morning and once in the evening; the mice in the control group were gavaged with an equal amount of normal saline for a total of 14 days. On the 15th day, the mice were weighed, anesthetized, and the eyeballs were removed for blood collection. The organs were dissected, weighed, and pathological examinations were performed. The blood routine was analyzed using an automatic blood cell analyzer, and the blood biochemical indicators were determined according to the instructions of the kit.

[0061] (5) Evaluation of anti-aging effects

[0062] ① Lifespan: The synchronized wild-type Caenorhabditis elegans N2 (hereinafter referred to as "nematodes") were cultured to the L4 stage, and then 30 nematodes were selected and transferred to a solid nematode growth medium (NGM) coated with a live E. coli OP50 bacterial solution (containing 25 mg / mL of the freeze-dried powder of the CPs product of the present invention). Abnormal nematodes were removed every day, and the surviving nematodes were transferred to new solid NGM, and the number of surviving nematodes was recorded until all died.

[0063] ② Reproduction: Pick one L4 nematode and transfer it to solid NGM coated with live E. coli OP50 solution (containing 25 mg / mL lyophilized powder of the CPs product of the present invention). Transfer the nematode to a new solid NGM every day and record the number of eggs laid by the nematode until the egg laying is completely completed.

[0064] ③ Antioxidant enzymes and malondialdehyde: L4 stage nematodes were transferred to NGM coated with Escherichia coli OP50 live bacterial solution (containing 25 mg / mL lyophilized powder of the CPs product of the present invention and 100 μM 5-fluorodeoxyuridine), and the nematodes were collected after culturing at 20°C for 12 days. The precipitate was taken out by centrifugation and weighed, and pre-cooled physiological saline was added and homogenized in an ice bath. After centrifugation, the supernatant was taken and the protein concentration, antioxidant enzyme (superoxide dismutase, catalase, glutathione S-transferase) activity and malondialdehyde content were determined according to the instructions of the kit.

[0065] ④ Others: L4 stage nematodes were transferred to NGM coated with E. coli OP50 live bacterial solution (containing 25 mg / mL lyophilized powder of the CPs product of the present invention), and the following indicators were tested after culturing at 20°C for 8 days:

[0066] Body shape: 10 nematodes were selected and transferred to a blank culture dish. After anesthesia with 1% (w / v) imidazole hydrochloride, the body length and width were observed using an inverted fluorescence microscope.

[0067] Feeding: Pick 10 nematodes and transfer them to solid NGM coated with live E. coli OP50 bacteria solution, and observe the contraction frequency of the nematode pharynx within 20 seconds.

[0068] Movement: Pick 10 nematodes and transfer them to blank solid NGM, and observe the number of head swings and body sinusoidal movements of the nematodes within 20 seconds.

[0069] Chemotaxis: Spread two equal-sized E. coli OP50 bacterial lawns on solid NGM, pick 30 nematodes and place them between the two bacterial lawns, and count the number of nematodes on the bacterial lawns after the nematodes are completely dispersed.

[0070] Lipofuscin: 25 nematodes were selected and transferred to a blank culture dish. After anesthesia with 1% (w / v) imidazole hydrochloride, the nematodes were observed under an inverted fluorescence microscope under an excitation light of 450-490 nm.

[0071] Reactive oxygen species: 25 nematodes were selected and transferred to a centrifuge tube. 100 μL of 10 μM DCFH-DA was added and the tube was kept in dark for 30 min. The nematodes were washed three times with M9 buffer and transferred to a black 96-well plate. The plates were anesthetized with 1% (w / v) imidazole hydrochloride and observed using an inverted fluorescence microscope.

[0072] Stress resistance: Pick 30 nematodes and transfer them to M9 buffer containing 10mM H2O2, and record the number of survival every 30min; pick 30 nematodes and transfer them to M9 buffer containing 500mM NaCl, and record the number of survival every 1h; pick 30 nematodes and transfer them to a 37℃ incubator, and record the number of survival every 1h.

[0073] Apoptosis: 25 nematodes were transferred to M9 buffer containing Escherichia coli OP50 and acridine orange (25 μg / mL) for staining in the dark for 1 h, then washed three times with M9 buffer, placed on solid NGM for recovery in the dark for 3 h, and then anesthetized with 1% (w / v) imidazole hydrochloride and observed under an inverted fluorescence microscope.

[0074] Epidermal barrier function: 25 nematodes were selected and transferred to a centrifuge tube, washed three times with M9 buffer, and the precipitate was collected after centrifugation. 100 μL Hoechst 33342 dye was added, and the tube was stained at 20°C in the dark for 15 min. The tube was centrifuged again and the supernatant was discarded. The tube was anesthetized with 1% (w / v) imidazole hydrochloride and observed under an inverted fluorescence microscope.

[0075] Test results:

[0076] (1) Microbiological and physical and chemical properties

[0077] The test results of the microbial and physicochemical properties of the CPs product of the present invention are shown in Table 2. The LP number of the product is 8.82×10 8 CFU / mL, KM quantity is 7.69×10 7 The content of volatile organic compounds in the beverage was 0.45%, which met the requirements of GB 7101-2022 National Food Safety Standard Beverages (alcohol content <0.50%). The volatile compounds were mainly alcohols, hydrocarbons, acids and esters, which were 4549.34, 1057.60, 472.57 and 31894.41 μg / L, respectively.

[0078] Table 2 Microbiological and physicochemical properties of CPs products of the present invention

[0079]

[0080] (2) Sensory quality

[0081] Table 3 shows the sensory evaluation results of the CPs product of the present invention. The color of the product is 82.56 points, dark purple, basically clear and transparent; the texture is 91.11 points, uniform and stable, without obvious precipitation or suspended matter; the sweetness and sourness are 71.75 and 80.38 points respectively, with moderate sweetness and sourness, harmonious taste, and no irritation; the astringency is 61.17 points, with a little astringency, but within an acceptable range, and the astringency may also improve the quality of the product and meet the preferences and tastes of some people (such as people who love red wine); the bitterness is 93.64 points, almost no bitterness; the fruity, mellow and fishy smells are 84.94, 77.16 and 95.62 points respectively, and the fruity, mellow and lactic acid fermentation flavors are coordinated and rich, without obvious odor or irritation; the comprehensive evaluation is 82.09 points. In addition, the emotional (preference) test score is 85.45 points, which has good consumer acceptance.

[0082] Table 3 Sensory evaluation of CPs products of the present invention

[0083]

[0084] (3) Food safety

[0085] Table 4 shows the results of the subacute oral toxicity test of the CPs product of the present invention. After continuous gavage of the product to mice for 14 days at 20 mL / kg·bw and twice / d (about 50 times the recommended intake for humans), no animal death or obvious abnormal behavior was observed, the food and water intake was good, the hair was smooth and shiny, and there was no significant difference in body weight and organ indexes (heart, lung, liver, kidney, spleen and ovary, etc.) compared with the control group; there was no significant difference in blood routine and liver and kidney function indicators compared with the control group, all within the normal reference value range; after dissection, there was no obvious pathological change in the appearance and structure of all organs, proving that the product has no subacute oral toxicity.

[0086] (4) Anti-aging effect

[0087] Table 5 shows the test results of the CPs product of the present invention for delaying aging in nematodes. Nematodes are a classic model organism for studying aging. Their genome, signaling pathways, age-related changes, etc. are highly conservative with humans. Any intervention measures that extend the lifespan of Caenorhabditis elegans may have a positive impact on human lifespan. The average and maximum lifespans of nematodes treated with this product were 15.53 and 21.67d, respectively, which were 28.67% and 25.04% higher than those of the control group, respectively. The body length (1.19mm), head swing and body sinusoidal movement frequency (40.32 and 14.18 / min) of nematodes treated with this product were significantly higher than those of the control group, indicating that it improved the body shape and exercise ability of nematodes. However, there were no significant differences in the swallowing frequency (131.95 / min), chemotaxis (14.05) or number of eggs laid (145.63) of nematodes treated with this product compared with the control group, indicating that it had no significant effect on the food intake or reproductive ability of nematodes, and its lifespan extension effect did not depend on calorie restriction, nor at the expense of reproductive ability.

[0088] Table 4 Effects of the CPs products of the present invention on the physiological and biochemical properties of mice

[0089]

[0090] Table 5 Effects of the CPs products of the present invention on the life span and physiological and biochemical characteristics of nematodes

[0091]

[0092] In addition, the superoxide dismutase, catalase and glutathione S-transferase activities (58.34, 8.50 and 37.31 U / mg prot) of nematodes treated with this product were significantly higher than those of the control group, while the levels of reactive oxygen, malondialdehyde and lipofuscin (51.96 RFU, 0.59 nmol / mg prot and 4.97 RFU) were significantly lower than those of the control group (Table 5), indicating that it enhanced the oxidative defense system of nematodes and alleviated oxidative stress in the body. Under oxidative, hyperosmotic and high temperature stress conditions, the average lifespan of nematodes treated with this product increased by 30.53%, 79.40% and 14.21% respectively compared with the control group, indicating that it enhanced the nematodes' ability to adapt or resist external environmental stimuli. The cell apoptosis of nematodes treated with this product decreased by 39.27%, and the epidermal barrier function increased by 24.26%, indicating that it inhibited the cell apoptosis of nematodes and improved the structural integrity and epidermal barrier function of the nematode epidermis.

[0093] In summary: In response to the common technical problems in the industry of poor sensory quality, low biological activity and many food additives of CPs products currently on the market, the present invention uses CPs as raw material, blueberry juice as a carrier, LP and KM as strains, and utilizes technologies such as probiotic metabolic transformation, flavor quantitative reconstruction and functional targeted regulation to exert the synergistic effect of multiple components to produce a fermented CPs product rich in probiotics and anthocyanins, with 0 added water and 0 additives, which has good physical and chemical characteristics, sensory quality, food safety and anti-aging effects.

[0094] Note: The above embodiments are only used to illustrate the present invention and are not intended to limit the technical solutions described in the present invention; therefore, although this specification has described the present invention in detail with reference to the above embodiments, a person skilled in the art should understand that the present invention can still be modified or replaced by equivalents; and all technical solutions and improvements thereof that do not depart from the spirit and scope of the present invention should be included in the scope of the claims of the present invention.

Claims

1. A probiotic fermentation production method for anti-aging collagen peptide products, characterized in that: Here are the steps: (1) Lactobacillus plantarum and Kluyveromyces marxianus were activated and expanded, respectively, and the precipitates were centrifuged after the culture; physiological saline was added to the precipitates for oscillation and washing, and the precipitates were centrifuged again after washing, and the obtained precipitates were recorded as LP bacterial sludge and KM bacterial sludge, respectively; (2) washing the blueberries, draining the water, and grinding to obtain blueberry pulp; then adding pectinase to the blueberry pulp, stirring continuously for enzymolysis, and centrifuging after enzymolysis to obtain the supernatant, which is the blueberry juice; (3) adding the collagen peptide to the blueberry juice prepared in step (2), stirring and dissolving to obtain a mixture of blueberry juice and collagen peptide, and heating and sterilizing the mixture; cooling the mixture after sterilization, and then adding the LP bacterial slurry prepared in step (1) after cooling, and performing a primary fermentation under the conditions of mechanical stirring and air flow; after the primary fermentation, adding the KM bacterial slurry prepared in step (1) again, and performing a secondary fermentation under the conditions of mechanical stirring and air flow; and after the secondary fermentation, post-ripening, sterilization, and filling are performed to obtain a collagen peptide product that delays aging.

2. The probiotic fermentation production method of an anti-aging collagen peptide product according to claim 1, characterized in that: The activation and expansion culture medium used for plant lactobacillus in step (1) is MRS medium, the inoculum size is 1-3%, and the culture temperature is 30-37°C; the activation culture time is 16-20h, and the expansion culture time is 8-12h; the activation and expansion culture medium used for Kluyveromyces marxianus is YM medium, the inoculum size is 1-3%, and the culture temperature is 22-28°C; the activation culture time is 12-16h, and the expansion culture time is 6-10h.

3. The probiotic fermentation production method of an anti-aging collagen peptide product according to claim 1, characterized in that: The temperature of the physiological saline in step (1) is 4-10°C, and the amount added is 3-5L of physiological saline per kg of precipitation; the equipment used for the centrifugation is an intermittent centrifuge, the centrifugal force is 5000-8000g, and the time is 10-20min.

4. The probiotic fermentation production method of an anti-aging collagen peptide product according to claim 1, characterized in that: The equipment used for grinding in step (2) is a colloid mill, the temperature of the colloid mill for treating blueberries is 5-15° C., and the time is 5-10 min; the equipment used for centrifugation is a tubular centrifuge, the centrifugal force is 15000-20000 g, and the time is 3-7 min; the amount of pectinase added is 0.1-0.2% (w / v, g / mL) of the blueberry pulp; the enzymatic hydrolysis time is 1-2 h, and the temperature is 45-55° C.

5. The probiotic fermentation production method of an anti-aging collagen peptide product according to claim 1, characterized in that: The amount of collagen peptide added in step (3) is 10-15% (w / v, g / mL) of the blueberry juice; the sterilization temperature is 70-90°C and the time is 20-30 min; and cooling refers to cooling to 30-37°C.

6. The probiotic fermentation production method of an anti-aging collagen peptide product according to claim 1, characterized in that: The inoculation amount of the LP bacterial sludge in step (3) is 0.1-0.2 g / L based on the volume of the mixture of blueberry juice and collagen peptide; the temperature of the first fermentation is 30-37° C., the time is 9-12 h; the stirring speed is 120-200 rpm, and the ventilation volume is 0.2-0.3 v / v·min.

7. The probiotic fermentation production method of an anti-aging collagen peptide product according to claim 1, characterized in that: The inoculation amount of the KM bacterial sludge in step (3) is 0.1-0.2 g / L based on the volume of the mixture of blueberry juice and collagen peptide; the temperature of the secondary fermentation is 22-28° C., the time is 10-15 h; the stirring speed is 120-200 rpm, and the ventilation volume is 0.1-0.2 v / v·min.

8. The probiotic fermentation production method of an anti-aging collagen peptide product according to claim 1, characterized in that: The post-ripening temperature in step (3) is 2-6°C for 6-12 hours; the sterilization method is UHT sterilization, the sterilization temperature is 130-140°C, and the sterilization time is 5-10 seconds.

9. The probiotic fermentation production method of an anti-aging collagen peptide product according to claim 1, characterized in that: The collagen peptides described in step (3) are commercially available food-grade collagen peptides, including fish-derived collagen peptides, pig-derived collagen peptides, bovine-derived collagen peptides and poultry-derived collagen peptides.

10. An anti-aging collagen peptide product prepared according to the method of any one of claims 1 to 9.

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