1. A bacillus coagulans BC2000 strain is preserved in China general microbiological culture Collection center (CGMCC) at 1 month and 13 months of 2021, and the preservation number is CGMCC No. 21621.

2. Use of bacillus coagulans BC2000 as claimed in claim 1 for probiotic nut production.

3. The use of claim 2, wherein the probiotic coating powder comprising bacillus coagulans BC2000 is prepared and then applied to the coating of the probiotic nuts.

4. The application according to claim 2, wherein the application specifically comprises:

(1) preparing probiotic coating powder: uniformly mixing bacillus coagulans BC2000, glucose, fermented yoghurt powder, milk mineral salt, malic acid, fructo-oligosaccharide and haematococcus pluvialis;

(2) preparing mucus: dissolving isomaltitol and maltodextrin with water to obtain coating mucus;

(3) coating probiotics and nuts: putting the nuts into coating equipment, starting the equipment, and coating;

(4) drying the probiotics nuts: and (4) baking the coated probiotic nuts in an oven.

5. The application of the probiotic coated powder as claimed in claim 4, wherein the probiotic coated powder comprises, by mass, 2-20 parts of Bacillus coagulans, 200-500 parts of glucose, 200-500 parts of fermented yoghurt powder, 3-30 parts of milk mineral salt, 2-20 parts of malic acid, 10-100 parts of fructo-oligosaccharide and 1-5 parts of Haematococcus pluvialis.

6. The use of claim 4, wherein the coating mucus comprises, by mass, 1-10 parts of isomalt, 20-40 parts of maltodextrin and 60-130 parts of water.

7. The use according to claim 5, wherein the preparation of the probiotic coating powder comprises: 2-20 parts of bacillus coagulans and 500 parts of 200-plus-500 parts of glucose are premixed to ensure that the bacillus coagulans and the glucose are uniformly mixed, and then the mixture is put into a three-dimensional mixer together with 200-plus-500 parts of fermented yoghurt powder, 3-30 parts of milk mineral salt, 2-20 parts of malic acid, 10-100 parts of fructo-oligosaccharide and 1-5 parts of haematococcus pluvialis for full mixing.

8. The use of any one of claims 2 to 5, wherein the probiotic nut is any one or more of: probiotic walnut kernels, probiotic almond kernels, probiotic cashew kernels and probiotic hazelnut kernels.

9. The use according to claim 5, wherein the dried fruit is any one or more of: dried cranberries, dried blackcurrant grapes and dried blueberries.

10. The application according to claim 2, characterized in that it is specifically:

(1) preparing probiotic coating powder: uniformly mixing bacillus coagulans BC2000, glucose, fermented yoghurt powder, milk mineral salt, malic acid, fructo-oligosaccharide and haematococcus pluvialis; wherein the coating powder is prepared by premixing 2-20 parts of bacillus coagulans and 500 parts of 200-containing milk sugar in an adding ratio to ensure that the bacillus coagulans and the 200-containing milk sugar are uniformly mixed, and then putting the obtained mixture, 200-containing milk acid milk powder, 3-30 parts of milk mineral salt, 2-20 parts of malic acid, 10-100 parts of fructo-oligosaccharide and 1-5 parts of haematococcus pluvialis into a three-dimensional mixer for fully mixing;

(2) preparing mucus: dissolving isomaltitol and maltodextrin with water to obtain coating mucus;

1-10 parts of isomaltitol and 20-40 parts of maltodextrin, and 60-130 parts of water are fully dissolved to prepare coating mucus

(3) Coating probiotics and nuts: putting the nuts into coating equipment, starting the equipment, and coating;

the coating equipment is divided into two sections, wherein the front end is a mucus adding end, and the rear end is a powder coating end; wherein the coating mucus is added into a front-end liquid storage tank of the coating device, the mucus adding parameter is 0.3-0.6 part of mucus, the time is 0.5-2min, the coating powder is added into a rear-end powder storage tank, the coating powder adding parameter is 0.7-1.5 parts of coating powder, and the time is 0.5-2 min;

(4) drying the probiotics nuts: baking the coated probiotic nuts in an oven;

(5) packaging probiotics and nuts: mixing and packaging the semi-finished product of the probiotics nut with dried fruit or other food;

wherein, the packaging is nitrogen-filled packaging, and meanwhile, the oxygen absorbent is added into the packaging bag.

Background

Nuts are a classification of closed fruit, with a hard peel containing 1 or more seeds. Such as walnut, cashew, etc., which are the essential parts of plants. The nuts respectively contain 36.0% of protein, 58.8% of fat, 72.6% of carbohydrate, vitamins (vitamin B, vitamin E, etc.), trace elements (phosphorus, calcium, zinc, iron), dietary fiber, etc. In addition, it also contains monounsaturated fatty acid and polyunsaturated fatty acid, including essential fatty acid of human body such as linolenic acid and linoleic acid. Has excellent effects on the growth and development of human bodies, the enhancement of physique and the prevention of diseases. According to the research of an authority, the following results are obtained: eating more than twice a week can reduce the risk of a person suffering from fatal heart disease, an accurate result of a U.S. physician health research program investigating twenty thousand men. However, the nut has the problems that the surface of the processed nut is easily oxidized and rancid, unpleasant flavor is generated, the product quality is influenced, and the nutritional value of the nut is reduced, and particularly the problem that the peeled walnut kernel is easily rancid in the quality guarantee period is prominent. In addition, the inability to decompose and utilize dietary fiber in nuts is also a problem.

Probiotics are living microorganisms which can benefit host bodies after being ingested in proper amount, and have the functions of adjusting intestinal flora and improving immunity. Probiotics have been reported for use in the processing of nuts.

For example, CN110521988A discloses a method for processing nut kernel powder rich in active microorganisms, which uses bacillus coagulans in the processing of nut kernel powder, wherein bacillus coagulans is a facultative anaerobe, which can form spores and produce lactic acid, and has the characteristics of acid resistance, heat resistance, salt resistance, and easy culture and preservation compared with common lactic acid bacteria. After entering a human body, the bacillus coagulans has higher tolerance to gastric acid and bile and can be adapted to the intestinal environment with low oxygen, and meanwhile, antibacterial substances such as L-lactic acid and the like are generated by fermentation, so that the pH value of the intestinal tract can be effectively reduced, harmful bacteria can be inhibited, and the growth and reproduction of beneficial bacteria such as bifidobacterium and the like can be promoted, thereby regulating the balance of microbial flora in the intestinal tract, improving the immunity of the organism and having a certain treatment effect on acute and chronic diarrhea, chronic constipation, abdominal distension and dyspepsia caused by flora imbalance. However, CN110521988A does not disclose the use of Bacillus coagulans strain, nor does it demonstrate the effectiveness of the use. In fact, it is worth noting that Bacillus coagulans, unlike other probiotics, is generally difficult to be found in the intestine in natural conditions due to the poor adherence of the bacteria to the intestinal epithelium. Therefore, the bacillus coagulans can only stay in the intestinal tract for a short time as a immigration in the intestinal tract, and after the bacillus coagulans is taken orally for a time of 4-7 days, the bacillus coagulans in the intestinal tract can be eliminated by defecation.

For another example, CN112335883A discloses a multi-component probiotic preparation, daily-absorbable nuts and a preparation method thereof, wherein bacillus coagulans, bifidobacterium, lactobacillus rhamnosus, lactobacillus acidophilus, lactobacillus paracasei and the like in specific proportions are used as main auxiliary materials, the auxiliary materials in specific proportions are compounded with prebiotics, probiotic factors, maltodextrin, fermented yoghurt powder and the like to prepare the multi-component probiotic preparation, and the probiotic preparation is added to the daily nuts by a specific multilayer coating process. The CN112335883A preparation contains two probiotic factors, namely resistant dextrin and isomaltulose, and the selected probiotics and the selected auxiliary materials are reasonably matched through creative experiments, so that the preparation has a good effect; meanwhile, the nuts prepared by the multi-element probiotic preparation have the characteristics of good digestion, easy absorption, good taste and the like. CN112335883A needs to be compounded with various probiotics, and whether the probiotic activity in the product can be maintained in a longer shelf life is not determined.

Thus, if a high-performance, multifunctional bacillus coagulans could be obtained, it would be beneficial to improve the performance of probiotic nut products; meanwhile, the strain which is simple in probiotic formula, resistant to nut processing technology, stable in activity and capable of effectively decomposing cellulose is developed and used for processing the probiotic nuts, so that the problems that the nuts are easy to oxidize and rancid in the quality guarantee period and the like are effectively solved, and the strain is a hotspot and a difficulty in the conventional nut processing.

Disclosure of Invention

[ problem to be solved ]

Aiming at least one of the following problems, the invention provides a multifunctional bacillus coagulans BC2000, which is used for nut processing, and develops a preparation and processing method of antioxidant probiotic nuts: (1) the performance of the bacillus coagulans needs to be improved; for example, most probiotics are not resistant to high temperature and are volatile, cannot withstand nut processing techniques, cannot maintain probiotic activity during the shelf life of the product, and cannot live to reach the intestinal tract of a consumer; (2) the surface of the processed nut is easily oxidized and rancid to generate unpleasant flavor, the product quality is influenced, the nutritional value of the nut is reduced, and particularly the problem that peeled walnut kernels are easily rancid in the quality guarantee period is obvious.

The bacillus coagulans BC2000 has the performances of resisting acid, cholate, producing lactic acid, inhibiting the growth of pathogenic bacteria, resisting high temperature and high pressure, secreting protease, amylase, lactase and the like, degrading nitrite, degrading various mycotoxins, producing plasmin at high yield and the like, and has wide application range and good use effect.

The processing method of the invention combines effective components and proportion with the process, can effectively solve the problems that the nuts are easy to oxidize and rancid in the quality guarantee period, the activity of probiotics keeps stable, and simultaneously can solve the problems that the probiotics can not resist the nut processing process and can not reach the intestinal tracts of consumers alive, and dietary fiber in the nuts can not be decomposed and utilized, and the like.

[ technical solution ] A

The first purpose of the invention is to provide a bacillus coagulans BC2000 which is preserved in China general microbiological culture Collection center of the culture Collection management Committee of microorganisms at 1 month and 13 months in 2021, wherein the preservation number is CGMCC No.21621, and the preservation address is No. 3 of No. 1 Xilu on Beijing.

The properties of the bacillus coagulans BC2000 are as follows:

(1) high temperature resistance: the high temperature resistance under the wet heat condition and the dry heat condition is excellent; wherein, the biological agent can keep certain survival under the condition of 160 ℃ dry heat, and the survival rate of the biological agent treated for 60min under the condition of 100 ℃ damp heat can reach more than 70 percent;

(2) and (3) bile salt resistance: after being treated for 24 hours by 0.3 percent of bile salt, the survival rate can be kept above 95 percent;

(3) acid resistance: after the treatment for 6 hours at the pH of 1.5, the survival rate can be kept more than 50 percent;

(4) storage stability: the survival rate can be kept at 96.92 percent after the tea is stored for 36 months in a dry and dark place;

(5) lactic acid production: the concentration of the fermentation supernatant after fermentation for 36 hours reaches 8.72 g/L;

(6) the enzyme is rich: can produce protease, lactase, amylase and plasmin at high yield; the amylase activity can reach 302.43U/mgprot, and the plasmin activity in the fermentation liquor can reach 467.9U/mL;

(7) degradable nitrite: 2.0mg/L of nitrite is degraded, and the degradation rate is up to 98% after 36 hours;

(8) inhibiting the growth of pathogenic bacteria: can inhibit growth of Escherichia coli, enterococcus faecalis, Streptococcus mutans, Staphylococcus aureus, and Salmonella typhi;

(9) can degrade various mycotoxins: can efficiently degrade zearalenone and can degrade 4 aflatoxins.

The second purpose of the invention is to provide the application of the multifunctional bacillus coagulans in the preparation of the probiotics nut.

The application is that probiotic coating powder containing bacillus coagulans BC2000 is prepared firstly, and then the probiotic coating powder is used for coating probiotic nuts.

The application according to claim 2, wherein the application specifically comprises:

preparing probiotic coating powder: uniformly mixing bacillus coagulans BC2000, glucose, fermented yoghurt powder, milk mineral salt, malic acid, fructo-oligosaccharide and haematococcus pluvialis;

preparing mucus: dissolving isomaltitol and maltodextrin with water to obtain coating mucus;

coating probiotics and nuts: putting the nuts into coating equipment, starting the equipment, and coating;

drying the probiotics nuts: and (4) baking the coated probiotic nuts in an oven.

In one embodiment of the invention, the use further comprises a probiotic nut packaging step; specifically, the semi-finished product of the probiotics and nuts obtained after baking is mixed with dried fruits or other foods for packaging.

In an embodiment of the present invention, the application specifically is:

the method comprises the following steps: preparing probiotic coating powder: uniformly mixing bacillus coagulans BC2000, glucose, fermented yoghurt powder, milk mineral salt, malic acid, fructo-oligosaccharide and haematococcus pluvialis.

Wherein the coating powder is added in a proportion of 2-20 parts (by weight, the same below) of bacillus coagulans (with spore content of 1 × 10)⁹-5×10⁹cfu/g) and 500 parts of 200-plus-500 parts of glucose are premixed to ensure that the two are uniformly mixed, and then the two are put into a three-dimensional mixer together with 500 parts of 200-plus-500 parts of fermented yoghurt powder, 3-30 parts of milk mineral salt, 2-20 parts of malic acid, 10-100 parts of fructo-oligosaccharide and 1-5 parts of haematococcus pluvialis for full mixing; the malic acid makes the environment of the bacillus coagulans to be acid, so that the bacillus coagulans can be protected from being inhibited by other bacterial colonies by inhibiting the growth of other microorganisms, wherein fructo-oligosaccharide is a prebiotic and can make the bacillus coagulans grow better during germination, so that the balance of human intestinal flora is adjusted, wherein haematococcus pluvialis is rich in astaxanthin, has strong oxidation resistance, can be attached to the surface of nuts, and delays the oxidative rancidity of the nuts; the bacillus coagulans BC2000 can generate cellulase after the human body germinates, and can decompose dietary cellulose in nuts, and mineral elements such as calcium, magnesium, iron, zinc and the like cannot be separated in the enzyme production process. The milk mineral salt is rich in calcium and zincThe nuts also contain mineral elements rich in iron, zinc, magnesium, etc. After the bacillus coagulans germinates, the bacillus coagulans can absorb various mineral elements to promote the bacillus coagulans to produce cellulase, meanwhile, various mineral elements can also activate the cellulase, improve the activity of the cellulase, promote the dietary cellulose in nuts to be decomposed into various sugars, and provide more nutrients for other probiotics in intestinal tracts. Various mineral elements can promote the propagation of the bacillus coagulans in the intestinal tract, so that free oxygen in the intestinal tract is consumed, the growth of anaerobic microorganisms and bifidobacteria in the intestinal tract is facilitated, and the balance of microbial flora in the intestinal tract is adjusted;

step two: preparing mucus: dissolving isomaltitol and maltodextrin in water to obtain coating mucus.

Wherein the coating mucus is prepared by fully dissolving 1-10 parts of isomaltitol and 20-40 parts of maltodextrin in 60-130 parts of water. Wherein maltodextrin is main adhesive, and has good solubility, no odor and high viscosity; wherein the isomaltitol is a new resource, has the functions of promoting the gastrointestinal peristalsis of a human body and softening the excrement, is a prebiotic, and can be matched with the bacillus coagulans to improve the survival rate of the bacillus coagulans in the human body; the mucilage has improved viscosity after the isomaltitol and the maltodextrin are prepared according to the proportion, ensures that the coating powder can be uniformly attached to the surfaces of the nuts, is not easy to fall off, and keeps the quantity of the bacillus coagulans in the product stable.

Step three: coating probiotics and nuts: putting the nuts into a coating device, starting the device, and coating.

The coating equipment is divided into two sections, wherein the front end is a mucus adding end, and the rear end is a powder coating end. Wherein the coating mucilage is added into a liquid storage tank at the front end of the coating device, the adding parameter of the mucilage is 0.3-0.6 part of the mucilage, the time is 0.5-2min, the coating powder is added into a powder storage tank at the rear end, the adding parameter of the coating powder is 0.7-1.5 parts of the coating powder, the time is 0.5-2min, and 10 parts of nuts are taken out of the device. Starting the equipment, and setting the rotating speed to be 30-50 Hz. Wherein the nuts are first placed in the mucilage adding end device and begin to automatically add mucilage. When the operation reaches the set time, the nuts with the coating mucilage are automatically transferred to the powder coating end, meanwhile, the powder coating end is automatically started to begin powder spraying and coating, and the set time is reached. Automatically transferring the wrapped semi-finished product into a baking tray. Wrap up in the clothing through this equipment and can make bacillus coagulans and haematococcus pluvialis evenly distributed on the nut surface, guarantee the stability of bacillus coagulans quantity in the product, can realize the automation of probiotic nut simultaneously, production efficiency is high, practices thrift the labour, reduction in production cost.

Step four: drying the probiotics nuts: and (4) baking the coated probiotic nuts in an oven.

Wherein, the baking is a staged baking. The baking parameters are as follows: the first stage baking temperature is 60 ℃, and the baking time is 1 h; the second stage baking temperature is 80 ℃, and the baking time is 1 h; the third stage baking temperature is 85 ℃, and the baking time is 1 h. The sectional baking can effectively ensure that the water in the product is slowly released, prevent the probiotics nut from being stiff in taste, keep crisp taste, reduce the water activity in the probiotics nut, and ensure that the bacillus coagulans is alive to reach the intestinal tract.

Step five: packaging probiotics and nuts: mixing the semi-finished product of the probiotics nut with dried fruit or other food, and packaging.

Wherein, the packaging is nitrogen-filled packaging, which can reduce the oxygen content in the packaging bag; meanwhile, an oxygen absorbent is added into the packaging bag to further reduce the oxygen content in the bag, and the oxygen absorbent and haematococcus pluvialis synergistically act to prevent the probiotic nuts from being oxidized and rancid within the shelf life to generate unpleasant flavor. Wherein still add the drier simultaneously in the wrapping bag, slow down the moisture migration between the material in the product to and prevent the moisture in the oxygen absorbent to migrate to the probiotic nut, guarantee that the taste of probiotic nut is crisp and reduce the water activity of probiotic nut, prevent the germination of bacillus coagulans, guarantee that bacillus coagulans can live and reach the intestinal.

[ beneficial effects of the invention ]:

compared with the prior art, the invention has the beneficial effects that:

(1) the Bacillus coagulans CGMCC No.21621 screened from the pickle has the characteristics of high temperature resistance, high pressure resistance, acid resistance and cholate resistance, has outstanding lactic acid producing capability, and can realize the industrial production of large batch with stable spore number of more than 1000 hundred million. The strain has outstanding high temperature resistance, can ensure that the strain still has stable viable count and unique acid resistance in a complex food processing environment, can germinate in an acid environment of about 5.2, is more acid-resistant than other strains in the market, and can have higher survival rate in a human gastric acid environment (about 2.0). In addition, the strain can inhibit the growth of pathogenic bacteria, can produce protease, amylase and lactase, can produce plasmin at high yield, and can degrade nitrite and various mycotoxins. The Bacillus coagulons CGMCC No.21621 of the invention is used for preparing the probiotic nuts, and not only endows the probiotic nuts with better quality, but also endows the probiotic nuts with higher safety.

(2) The Bacillus coagulousns CGMCC No.21621 of the invention can keep the activity of Bacillus coagulans in the shelf life of the probiotic nuts; can pass through gastric acid and choline and germinate after reaching the intestinal tract of a consumer; germinates into a nutrient body, can secrete protease to promote the digestion of protein into small molecules, and is more beneficial to the absorption of a human body; the cellulase can be secreted to promote digestion and decomposition of dietary cellulose in nuts into polysaccharide, and can provide nutrition for other probiotics in intestinal tracts and human bodies; can germinate in the intestinal tract, secrete acidic substances to reduce Ph in the intestinal tract and inhibit the propagation of putrefying bacteria; fructo-oligosaccharide and isomaltitol can not be digested and absorbed by human body, so that intestinal peristalsis can be increased, and diarrhea and constipation can be effectively relieved under the synergistic effect of fructo-oligosaccharide and isomaltitol; germination and proliferation in intestinal tracts, consumption of free oxygen in the intestinal tracts and improvement of the growth environment of anaerobic probiotics, so that the intestinal flora can be adjusted; in addition, the pH in the intestinal tract can be reduced after germination, so that the environment in the intestinal tract is more beneficial to beneficial bacteria; meanwhile, various enzymes can be secreted to decompose protein and dietary cellulose and provide micromolecular protein and polysaccharide, so that the immunity of the human body is improved.

(3) When the Bacillus coagulans CGMCC No.21621 is applied to the preparation of probiotic nuts, the Bacillus coagulans CGMCC No.21621 is matched with haematococcus pluvialis for use. Haematococcus pluvialis is a freshwater unicellular green alga belonging to the phylum Chlorophyta, the order Volvocales, the family Haematococcus, the genus Haematococcus. The algae can accumulate a large amount of astaxanthin to show red color, so it is called Haematococcus pluvialis, also called Haematococcus pluvialis. Haematococcus is an algae food which is discovered by the scientific community at present and is rich in nutritional value and medicinal value after spirulina and chlorella. Haematococcus pluvialis is a unicellular green alga that grows mainly in fresh water, and is rarely distributed in the ocean, but salinity in seawater is favorable for accumulation of astaxanthin in algal bodies. Haematococcus pluvialis is a recognized ideal source for producing natural astaxanthin in the nature, and astaxanthin, which is the most efficient pure natural antioxidant discovered at present, shows good biological activity in the aspects of free radical scavenging, anti-aging, anti-tumor, immunoregulation and the like, and is widely applied to the fields of functional foods, medicines and cosmetics.

(4) When the Bacillus coagulans CGMCC No.21621 is applied to the preparation of probiotic nuts, the Bacillus coagulans CGMCC No.21621 is matched with isomaltulose for use. Isomalt is a functional sugar alcohol which is emerging internationally in recent years and is an excellent substitute for sucrose, starch sugar and other sugar alcohols. The sweetness is 50-60% of that of cane sugar, and the sugar-free cane sugar has the characteristics of low hygroscopicity, high stability, high tolerance, low calorie, pure sweetness and the like. The product is extremely safe, and the FDA in the United states gives the product a GRAS (generally recognized as safe) status, and the daily intake of the product is not limited. From the nutritional point of view, isomalt is a carbohydrate, and from the physiological point of view, isomalt is not easily desorbed and absorbed in the human body and is not decomposed and utilized by most microorganisms. According to "notice of approval of new resource food such as galactooligosaccharide by Ministry of health" (No. 20 in Notification by Ministry of health 2008), isomalt belongs to a new food raw material. The application range of the food is suitable for various foods, but does not include infant foods. And the consumption is less than or equal to 100 g/day. And the isomaltitol is an excellent bifidobacterium proliferation factor, although the isomaltitol can not be utilized by the human body and enzyme systems of most microorganisms, the isomaltitol can be decomposed and utilized by the bifidobacteria in the intestinal tract of the human body, thereby promoting the growth and the propagation of the bifidobacteria, maintaining the microecological balance of the intestinal tract and being beneficial to the health of the human body.

(5) According to the process for preparing the probiotics nut, the surfaces of the probiotics processed and produced by the process are uniformly coated with the powder, the powder is attractive, the powder is not prone to falling off, and hands are not dirty when the probiotics nut is eaten. The coating powder on the surface of the probiotics nut forms a protective layer, oxygen can be prevented from contacting with the surface of the nut, meanwhile, the haematococcus pluvialis and nitrogen-filled package and oxygen absorbent synergistic effect in the probiotics nut can delay the nut oxidation rancidity, the nut surface oxidation rancidity is reduced, and the quality of the probiotics nut is guaranteed.

[ biological Material Collection ]

The Bacillus coagulans BC2000 is classified and named as Bacillus coagulans, is preserved in China general microbiological culture Collection center (CGMCC) No.21621 in 13 months at 2021, and has the preservation address of No. 3 North Chen West Lu No. 1 of the sunward area in Beijing.

Detailed Description

The invention is further described with reference to specific examples.

Experimental materials and equipment: the culture medium components (such as yeast extract, beef extract, peptone, sodium bicarbonate, potassium dihydrogen phosphate, magnesium sulfate heptahydrate, manganese sulfate, calcium chloride and the like), the probiotic nut components (such as glucose, fermented yoghurt powder, milk mineral salt, malic acid, fructo-oligosaccharide, haematococcus pluvialis, isomaltitol, maltodextrin and the like) are common commercial products, and can be purchased through conventional commercial channels, such as a platform such as arbibaba 1688 and the like or other common biochemical reagent purchasing platforms and the like.

Example 1 screening and Performance determination of Bacillus coagulans

1 screening, identification and preservation of strains

(1) Firstly, crushing pickled pickles, adding 15mL of sterile water, violently shaking for 20min, and then carrying out water bath at 80 ℃ for 20 min; then 100uL of the diluent is taken and coated on an agar medium plate (the formula of the medium is 10.0g/L of yeast extract, 10.0g/L of beef extract, 10.0g/L of peptone, 1.5g/L of sodium bicarbonate, 1.5g/L of monopotassium phosphate, 1.0g/L of magnesium sulfate heptahydrate, 0.1g/L of manganese sulfate, 1.0g/L of calcium chloride and 1L of deionized water) by adopting a concentration gradient dilution method, the medium plate is cultured for 48 hours at 45 ℃, and strains with large bacterial colonies and fast growth are selected and further streaked, separated and purified.

(2) The separated and purified strains obtained in the last step are preserved by a glycerin tube; then inoculating the bacterial liquid into MRS culture medium, and culturing until the thallus concentration is 10⁸cfu/mL, then at a final concentration of 10⁷The addition amount of cfu/mL was added to a solution containing 0.5% bile salt, incubated at 37 ℃ for 24 hours, and plated on agar medium plates to determine the survival rate. And selecting strains with the survival rate of more than 95 percent for further screening.

(3) The strain was treated at 37 ℃ for 2 hours in simulated gastric fluid (pH 3.0) and plated on agar medium plates to determine the survival rate. Obtaining 29 strains of the strain with the survival rate of more than 70 percent.

(4) And (3) enzyme activity determination: respectively inoculating single bacillus coagulans colonies obtained in the last step into an MRS seed culture medium, culturing at 37 ℃ for 36h, transferring to a fermentation culture medium, and culturing at 37 ℃ for 48h at 200r/min to obtain fermentation liquor; taking the fermentation supernatant, adding 60% ammonium sulfate powder into the crude enzyme solution under ice bath condition, and precipitating at 4 ℃ to obtain protein in the crude enzyme solution to obtain precipitate; re-dissolving the obtained precipitate with Tris-HCl buffer solution, and dialyzing at 4 ℃ to obtain concentrated enzyme solution; the amylase activity, the lactase activity and the protease activity of 29 strains of bacteria were respectively measured by using an amylase kit (purchased from Nanjing institute of bioengineering, cat # C016-1-1, iodine-starch colorimetric method), a lactase kit (purchased from Nanjing institute of bioengineering, cat # A082-1, for measuring lactase) and a protease kit (purchased from Shanghai Haoling Biotech Co., Ltd., cat # HL 15021.2). Obtaining 15 strains with better comprehensive performance.

(5) Activating the single bacillus coagulans colony obtained in the last step, preparing a seed culture solution, performing fermentation culture for 28 hours to obtain a fermentation culture solution, and centrifuging at 10000rpm for 5min to obtain a fermentation supernatant. And (3) mixing the fermentation supernatant with a standard product of zearalenone (or aflatoxin) and measuring the degradation effect of zearalenone (or aflatoxin). The strain with the best performance is obtained and named as Bacillus coaguluns BC 2000.

(6) The Bacillus coagulons BC2000 is subjected to taxonomic molecular identification by adopting 16SrDNA sequencing, the sequencing result is subjected to Blast comparison in NCBI, the similarity between the 16SrDNA base sequence and the Bacillus (Bacillus coagulons) is found to be the highest, and the Bacillus coagulans is determined to be identified. Bacillus coagulans BC2000 is preserved in China general microbiological culture Collection center (CGMCC) at 1 month and 13 months in 2021, the preservation number is CGMCC No.21621, and the preservation address is No. 3 Siro No. 1 Chen of the rising area in Beijing.

2. Culture of strain and preparation of microbial inoculum

Preparation of liquid microbial inoculum

The method comprises the following steps: inoculating single colony of Bacillus coagulans CGMCC No.21621 in MRS seed culture medium, culturing at 37 deg.C for 36 hr, transferring to fermentation culture medium, culturing at 37 deg.C for 40-56 hr at 200r/min to obtain strain with number of 1.0 × 10⁸-5.0×10⁹CFU/mL fermentation broth.

The second method comprises the following steps:

(1) slant thallus activation

Streaking and inoculating on slant culture medium (composed of peptone 10, beef extract 3, NaCl 5, bran 10, agar 15-20, pH 7.0-7.2, in g/L), and culturing at 30 deg.C for 24-48 hr;

(2) shake flask and seed tank culture

Scraping and washing mature bacterial mud on the inclined plane by using sterile water, putting the bacterial mud into a sterile triangular flask with glass beads inside to oscillate and disperse the bacterial mud to obtain uniform bacterial suspension, heating the bacterial suspension in a water bath at 80 ℃ for 10 minutes, and inoculating the bacterial suspension into the triangular flask for shaking culture in an inoculation amount of 1-10% of the volume ratio; the culture conditions were: the rotating speed of the shake flask is 200 r/min; the stirring speed of the seeding tank is 180r/min, and the ventilation volume is 1m³The culture temperature is 40 ℃, and the culture time is 24 h; the composition of the culture medium is as follows in g/L: 10 parts of bran, 8 parts of yeast extract, 8 parts of soybean meal and K parts of₂HPO₄ 3，NaCl 5，MnSO₄·H₂O 0.3，pH 7.0；

(3) Cultivation in fermenter

Inoculating the seed culture solution into a fermentation tank in an inoculation amount of 2% by volume; the fermentation medium consists of a carbon source, a nitrogen source and inorganic salts, and the fermentation medium comprises the following components in g/L: glucose 10, peptone 5-20, K₂HPO₄ 3，NaCl 5，MnSO₄·H₂0.3 of O, 2 to 10 of calcium carbonate and 7.0 of pH; the culture conditions of the fermentation tank are as follows: stirring speed of 200r/min and air flow of 1m³The culture temperature is 40 ℃, and the culture time is 18-48 h; defoaming according to the rising condition of foam in the culture process; taking the fermentation liquor for microscopic examination, and placing in a tank to finish the culture when more than 90% of the thalli form spores.

Preparation of B solid microbial inoculum

Centrifuging Bacillus coagulans CGMCC No.21621 bacteria solution cultured in fermentation tank, mixing spore and dry starch at weight ratio of 1:2, drying at 40-50 deg.C for 20-24 hr, pulverizing, sieving, adding adjuvants maltodextrin, mixing, and making into microecological preparation with spore content of 1 × 10⁹cfu/g or more.

3. Bacillus coagulans BC2000 performance measurement

(1) High temperature resistance

High temperature resistance under dry heat conditions: and uniformly spreading 100g of the prepared solid microbial inoculum powder in a tray, placing the tray in a high-temperature drying box with different temperatures for baking for a certain time, taking out the tray, placing the tray in a dryer for naturally cooling to room temperature, measuring the number of spores by adopting a plate coating method through gradient dilution, and calculating the survival rate (the survival rate is the number of the treated spores/the initial number of the spores). The results are shown in Table 1.

High temperature resistance under humid and hot conditions: and (4) treating the liquid microbial inoculum prepared by the second method for a period of time at different water bath temperatures, counting spores, and calculating the survival rate. The results are shown in Table 2.

The result shows that Bacillus coagulans CGMCC No.21621 can keep certain survival after being treated for 60min under the dry-heat condition of 160 ℃, the survival rate of the Bacillus coagulans CGMCC No.21621 can reach more than 70 percent after being treated for 60min under the damp-heat condition of 100 ℃, and the heat resistance of the Bacillus coagulans CGMCC No.21621 is very good.

TABLE 1

TABLE 2

(2) Resistance to bile salts

In order to determine the spore survival rate of the bacillus coagulans CGMCC No.21621 spores under different bile salt concentrations, 0.03%, 0.1%, 0.2% and 0.3% sodium taurocholate are respectively added to obtain an MRS culture medium, and the MRS culture medium is placed at 37 ℃ for 24h and then the spores are counted. The results are shown in Table 3.

TABLE 3

Bile salt concentration	0.03％	0.10％	0.20％	0.30％
					Spore survival rate	100％	100％	98.24％	94.94％

(3) Acid resistance

Adding proper amount of Bacillus coagulans CGMCC No.21621 liquid microbial inoculum prepared by the second method into MRS culture medium which is adjusted to pH of 1.5, 2.5, 3.5 and 4.5 by 0.1mol/l hydrochloric acid, treating at 37 ℃ for a period of time, and determining the survival rate. The results show that the survival rates after treatment at 37 ℃ for 2h or 6h are shown in Table 4. The acid resistance of the bacillus coagulans CGMCC No.21621 is excellent.

TABLE 4

Different conditions	pH 4.5	pH 3.5	pH 2.5	pH 1.5
					Survival rate after 2h	100.00％	99.88％	96.29％	84.93％
Survival rate after 6h	95.28％	86.24％	77.36％	54.39％

(4) Storage stability Properties

The solid microbial inoculum prepared by the method is placed in a dry and dark place and is stored at room temperature. Spore survival rates were calculated by performing assays at 1, 2, 3, 4, 5, 6, 8, 12, 16, 20, 24 and 36 months, respectively. The results are shown in Table 5.

TABLE 5

Time of detection

1 month

2 months old

3 months old

4 months old

6 months old

8 months old

12 months old

16 months old

24 months

36 months old

Survival rate

100.00％

100.00％

100.00％

99.99％

99.89％

99.59％

98.78％

98.42％

98.02％

96.92％

(5) Lactic acid producing, enzyme producing and nitrite degrading properties

Activating Bacillus coagulans CGMCC No.21621 to prepare a seed culture solution, and then inoculating the seed culture solution into a fermentation tank according to the inoculation amount of 2% of the volume ratio; fermentation medium, in g/L: glucose 10, peptone 5-20, K₂HPO₄ 3，NaCl 5，MnSO₄·H₂0.3 of O, 2 to 10 of calcium carbonate and 7.0 of pH; the culture conditions of the fermentation tank are as follows: stirring speed of 200r/min and air flow of 1m³The culture temperature is 40 ℃, and the culture time is 36 h.

And after the fermentation culture is finished, centrifuging at 10000rpm for 5min, taking the fermentation supernatant, and measuring the concentration of lactic acid in the fermentation supernatant to be 8.72 g/L.

Cooling the fermented supernatant, adding 60% ammonium sulfate powder under ice bath condition, and precipitating at 4 deg.C to obtain protein in the crude enzyme solution to obtain precipitate; and re-dissolving the obtained precipitate with Tris-HCl buffer solution, and dialyzing at 4 ℃ to obtain concentrated enzyme solution. The activity of amylase, lactase and protease obtained by measuring the concentrated enzyme solution (the kit purchased from Nanjing institute of bioengineering is adopted, and the product numbers are C016-1-1, A082-1 and HL15021.2 respectively), and the results show that the activity of the amylase can reach 302.43U/mgprot, the activity of the lactose is 28.56U/mgprot, and the activity of the protease is 1.28U/mg/min. Wherein U/mgprot represents hydrolysis of 10mg starch or 1nmol lactose per minute per mg tissue protein and is defined as 1 enzyme activity unit; u/mg/min represents an increasing change of 0.01 absorbance per minute per unit enzyme.

The plasmin activity in the fermentation supernatant was determined by reference to Astrup et al, showing a plasmin activity up to 467.9U/mL.

Determination of nitrite degradation capability: transferring the bacteria liquid after fermentation culture to an MRS culture medium added with nitrite with the initial concentration of 2.0mg/L according to the inoculation amount of 1 percent, taking 1mL of fermentation liquid after 0, 24, 36 and 48 hours respectively, and centrifuging. Sucking 0.4mL of supernatant into a colorimetric tube, adding distilled water to 5mL, adding 0.2mL of color developing agent mixed by A, B equal volumes of Grignard reagent, mixing uniformly, and heating in boiling water bath for 1 min. The absorbance was measured at 524nm wavelength and the blank tube was zeroed. And calculating the concentration of sodium nitrite in the fermentation liquor according to a standard curve, and respectively measuring the residual concentration of the sodium nitrite after 24 hours and 36 hours to calculate the degradation rate, wherein the results show that the degradation rates of 24 hours and 36 hours reach 88.5 percent and 98.2 percent.

(6) Pathogenic bacteria inhibiting properties

Inoculating 100 mu L of Bacillus coagulans CGMCC No.21621 bacterial liquid into 10mL of MRS liquid culture medium, and performing shake culture at 40 ℃ for 24h at 100rpm to obtain an indication bacterial liquid for later use. Respectively inoculating 100 μ L of pathogenic bacterial liquid (Escherichia coli, enterococcus faecalis, Streptococcus mutans, Staphylococcus aureus, and Salmonella typhi) into 10mL of liquid LB culture medium, and shake culturing at 37 deg.C for 24 hr at 100rpm to obtain pathogenic bacterial liquid for use. Diluting the pathogenic bacteria liquid cultured to logarithmic phase to 10%⁵And (3) uniformly coating 100 mu L of the bacterial suspension on an MRS solid culture medium, erecting sterile oxford cups on the MRS solid culture medium coated with the pathogenic bacterium liquid by using forceps, adding 200 mu L of indicator bacterium liquid into each oxford cup, taking 200 mu L of sterile water as a blank control, placing 4 oxford cups on each plate, arranging 3 parallel oxford cups on each plate, using one control, slightly covering the plates, placing the plates in a 37-DEG C constant-temperature incubator, culturing for 48 hours, and observing. The result shows that Bacillus coagulans CGMCC No.21621 has inhibition effect on the pathogenic bacteria, and obvious inhibition zones appear on the plate.

(7) Mycotoxin degrading performance

Activating Bacillus coagulans CGMCC No.21621 to prepare a seed culture solution, and then inoculating the seed culture solution into a fermentation tank according to the inoculation amount of 2% of the volume ratio; fermentation medium, in g/L: glucose 10, peptone 5-20, K₂HPO₄ 3，NaCl 5，MnSO₄·H₂0.3 of O, 2 to 10 of calcium carbonate and 7.0 of pH; the culture conditions of the fermentation tank are as follows: stirring speed of 200r/min and air flow of 1m³The culture temperature is 40 ℃, and the culture time is 36 h. After the fermentation culture is finished, obtaining a fermentation culture solution; centrifuging the fermentation culture solution at 10000rpm for 5min to obtain fermentation supernatant.

The application of degrading zearalenone comprises the following steps: mixing 900 μ L of the fermentation supernatant with 100 μ L of Zearalenone (ZEA) standard to a final ZEA standard concentration of 50 μ g/mL; meanwhile, using a medium containing the same final concentration of ZEA as a control, samples were taken at reactions 0h, 1h, 2h, 4h, 6h, and 8h, respectively, and the ZEA concentration was measured, and the degradation rate of ZEA (ZEA degradation rate (%) — (initial ZEA concentration-measured ZEA concentration after sampling)/initial ZEA concentration × 100%) was calculated. The results show that the degradation rates of the ZEA after 1h, 2h and 4h are 95.2%, 98.6% and 100% respectively.

The application of the aflatoxin degradation: crushing a peanut meal sample containing aflatoxin, sieving with a 40-mesh sieve, uniformly mixing, sampling by adopting a GB/T14699.1-2005 method, weighing 5.00g of the sample, placing the sample in a glass container, sealing a four-layer gauze newspaper, and sterilizing at 121 ℃ for 20min in a high-temperature high-pressure sterilization pot. Transferring the sterilized peanut meal to a fermentation vessel, adding 1mL of fermentation culture solution of Bacillus coagulousns CGMCC No.21621 into the treated sample, adding a proper amount of sterile distilled water, stirring and mixing uniformly, performing constant-temperature solid fermentation at 38 ℃, and fermenting for 48 hours to obtain a sample after removing four aflatoxins after solid fermentation. Collecting fermented samples, drying, respectively introducing each part of peanut meal into a centrifugal tube, adding acetonitrile-water solution (70+30) for extraction, washing a fermentation vessel, and introducing into the centrifugal tube together, so as to avoid the influence of uneven distribution of the toxin in the meal on the experimental result; mixing, ultrasonic treating for 10min, oscillating for 30min, centrifuging at 8000r/min for 10min, collecting the filtered supernatant, purifying with solid phase purification column, collecting the liquid, freeze concentrating, re-dissolving with methanol-water solution (50+50) in the filter membrane, and detecting with LC-MS/MS. Chromatographic conditions are as follows: a chromatographic column: BEH C18 column (2.1 mm. times.100 mm, 1.7 μm) mobile phase A was acetonitrile, B was ammonium formate solution, column temperature: 45 ℃, flow rate: 0.3mL/min, sample size: 5 mu L of the solution; the method is adopted to detect the residual quantity of the four toxins in the sample after the aflatoxin removal, and the corresponding removal rate of the four aflatoxins is calculated. The results show that the removal rates of aflatoxins AFTB1, AFTB2, AFTG1 and AFTG2 are respectively: 72.4%, 39.3%, 52.82%, 56.76%.

Example 2 application of multifunctional Bacillus coagulosns BC2000 to probiotic nut production

The method for applying the multifunctional Bacillus coagulosans BC2000 to the production of the probiotics nuts comprises the following steps:

(1) preparing probiotic coating powder:

5 parts of bacillus coagulans (the spore content reaches 1 multiplied by 10)⁹More than cfu/g, calculated according to the mass parts, the following are the same) and 350 parts of glucose powder are premixed to ensure that the two are uniformly mixed, and then the mixture is put into a three-dimensional mixer together with 350 parts of fermented yoghurt powder, 11 parts of malic acid, 16 parts of milk mineral salt, 55 parts of fructo-oligosaccharide and 3 parts of haematococcus pluvialis for full mixing;

(2) preparing mucus:

preparing coating liquid from 5 parts of isomalt, 30 parts of maltodextrin and 90 parts of water;

(3) probiotics nut coating

Putting 10 parts of peeled walnut kernels into a coating device, and putting mucilage into a liquid storage tank at a mucilage adding end of the coating device, wherein the adding parameters of the mucilage are as follows: 0.4 part of mucus, and the time is 1min, the coating powder is put into a powder storage tank at the powder coating end, and the coating powder adding parameters are as follows: coating powder 1 part for 1min, setting the rotating speed at 40Hz, and starting the equipment. Transferring the nuts with the mucilage in the mucilage adding end to the powder coating end after 1min, simultaneously starting automatic adding of the coating powder, and transferring the coated semi-finished product to a baking tray after 1 min.

(4) Probiotic nut drying

And putting the semi-finished products in the baking tray into an oven for staged baking. The baking parameters are as follows: the first stage baking temperature is 60 ℃, and the baking time is 1 h; the second stage baking temperature is 80 ℃, and the baking time is 1 h; the third stage baking temperature is 85 ℃, and the baking time is 1 h.

(5) Probiotic nut packaging

Mixing semi-finished products of probiotic nuts (probiotic semen Juglandis, probiotic almond kernel, probiotic cashew kernel, probiotic hazelnut kernel) and dried fruits (cranberry, black currant raisin, and dried blueberry), packaging with nitrogen, and adding oxygen absorbent and desiccant.

Example 3

(1) Preparing probiotic coating powder:

weighing 2 parts of bacillus coagulans and 350 parts of glucose powder, premixing, uniformly mixing, and putting the mixture, 350 parts of fermented yoghurt powder, 11 parts of malic acid, 16 parts of milk mineral salt, 55 parts of fructo-oligosaccharide and 3 parts of haematococcus pluvialis into a three-dimensional mixer for fully mixing;

(2) preparing mucus:

preparing coating liquid from 5 parts of isomalt, 30 parts of maltodextrin and 90 parts of water;

(3) probiotics nut coating

Putting 10 parts of peeled walnut kernels into a coating device, and putting mucilage into a liquid storage tank at a mucilage adding end of the coating device, wherein the adding parameters of the mucilage are as follows: 0.4 part of mucus, and the time is 1min, the coating powder is put into a powder storage tank at the powder coating end, and the coating powder adding parameters are as follows: coating powder 1 part for 1min, setting the rotating speed at 40Hz, and starting the equipment. Transferring the nuts with the mucilage in the mucilage adding end to the powder coating end after 1min, simultaneously starting automatic adding of the coating powder, and transferring the coated semi-finished product to a baking tray after 1 min.

(4) Probiotic nut drying

And putting the semi-finished products in the baking tray into an oven for staged baking. The baking parameters are as follows: the first stage baking temperature is 60 ℃, and the baking time is 1 h; the second stage baking temperature is 80 ℃, and the baking time is 1 h; the third stage baking temperature is 85 ℃, and the baking time is 1 h.

(5) Probiotic nut packaging

Mixing semi-finished products of probiotic nuts (probiotic semen Juglandis, probiotic almond kernel, probiotic cashew kernel, probiotic hazelnut kernel) and dried fruits (cranberry, black currant raisin, and dried blueberry), packaging with nitrogen, and adding oxygen absorbent and desiccant.

Example 4

(1) Preparing probiotic coating powder:

weighing 20 parts of bacillus coagulans, premixing with 350 parts of glucose powder to ensure that the bacillus coagulans and the glucose powder are uniformly mixed, and then putting the bacillus coagulans, the malic acid, the 16 parts of milk mineral salt, 55 parts of fructo-oligosaccharide and 3 parts of haematococcus pluvialis into a three-dimensional mixer for fully mixing;

(2) preparing mucus:

preparing coating liquid from 5 parts of isomalt, 30 parts of maltodextrin and 90 parts of water;

(3) probiotics nut coating

Putting 10 parts of peeled walnut kernels into a coating device, and putting mucilage into a liquid storage tank at a mucilage adding end of the coating device, wherein the adding parameters of the mucilage are as follows: 0.4 part of mucus, and the time is 1min, the coating powder is put into a powder storage tank at the powder coating end, and the coating powder adding parameters are as follows: coating powder 1 part for 1min, setting the rotating speed at 40Hz, and starting the equipment. Transferring the nuts with the mucilage in the mucilage adding end to the powder coating end after 1min, simultaneously starting automatic adding of the coating powder, and transferring the coated semi-finished product to a baking tray after 1 min.

(4) Probiotic nut drying

And putting the semi-finished products in the baking tray into an oven for staged baking. The baking parameters are as follows: the first stage baking temperature is 60 ℃, and the baking time is 1 h; the second stage baking temperature is 80 ℃, and the baking time is 1 h; the third stage baking temperature is 85 ℃, and the baking time is 1 h.

(5) Probiotic nut packaging

Mixing semi-finished products of probiotic nuts (probiotic semen Juglandis, probiotic almond kernel, probiotic cashew kernel, probiotic hazelnut kernel) and dried fruits (cranberry, black currant raisin, and dried blueberry), packaging with nitrogen, and adding oxygen absorbent and desiccant.

Comparative example 1

(1) Preparing probiotic coating powder:

weighing 5 parts of maltodextrin and 350 parts of glucose powder, premixing, uniformly mixing, and putting the mixture, 350 parts of fermented yoghurt powder, 11 parts of malic acid, 16 parts of milk mineral salt, 55 parts of fructo-oligosaccharide and 3 parts of haematococcus pluvialis into a three-dimensional mixer for fully mixing;

(2) preparing mucus:

preparing coating liquid from 5 parts of isomalt, 30 parts of maltodextrin and 90 parts of water;

(3) probiotics nut coating

Putting 10 parts of peeled walnut kernels into a coating device, and putting mucilage into a liquid storage tank at a mucilage adding end of the coating device, wherein the adding parameters of the mucilage are as follows: 0.4 part of mucus, and the time is 1min, the coating powder is put into a powder storage tank at the powder coating end, and the coating powder adding parameters are as follows: coating powder 1 part for 1min, setting the rotating speed at 40Hz, and starting the equipment. Transferring the nuts with the mucilage in the mucilage adding end to the powder coating end after 1min, simultaneously starting automatic adding of the coating powder, and transferring the coated semi-finished product to a baking tray after 1 min.

(4) Probiotic nut drying

And putting the semi-finished products in the baking tray into an oven for staged baking. The baking parameters are as follows: the first stage baking temperature is 60 ℃, and the baking time is 1 h; the second stage baking temperature is 80 ℃, and the baking time is 1 h; the third stage baking temperature is 85 ℃, and the baking time is 1 h.

(5) Probiotic nut packaging

Mixing semi-finished products of probiotic nuts (probiotic semen Juglandis, probiotic almond kernel, probiotic cashew kernel, probiotic hazelnut kernel) and dried fruits (cranberry, black currant raisin, and dried blueberry), packaging with nitrogen, and adding oxygen absorbent and desiccant.

Comparative example 2

(1) Preparing probiotic coating powder:

weighing 5 parts of bacillus coagulans and 350 parts of glucose powder, premixing, uniformly mixing, and then putting the bacillus coagulans and the glucose powder together with 350 parts of fermented yoghurt powder, 11 parts of malic acid, 16 parts of maltodextrin, 55 parts of fructo-oligosaccharide and 3 parts of haematococcus pluvialis into a three-dimensional mixer for fully mixing;

(2) preparing mucus:

preparing coating liquid from 5 parts of isomalt, 30 parts of maltodextrin and 90 parts of water;

(3) probiotics nut coating

Putting 10 parts of peeled walnut kernels into a coating device, and putting mucilage into a liquid storage tank at a mucilage adding end of the coating device, wherein the adding parameters of the mucilage are as follows: 0.4 part of mucus, and the time is 1min, the coating powder is put into a powder storage tank at the powder coating end, and the coating powder adding parameters are as follows: coating powder 1 part for 1min, setting the rotating speed at 40Hz, and starting the equipment. Transferring the nuts with the mucilage in the mucilage adding end to the powder coating end after 1min, simultaneously starting automatic adding of the coating powder, and transferring the coated semi-finished product to a baking tray after 1 min.

(4) Probiotic nut drying

And putting the semi-finished products in the baking tray into an oven for staged baking. The baking parameters are as follows: the first stage baking temperature is 60 ℃, and the baking time is 1 h; the second stage baking temperature is 80 ℃, and the baking time is 1 h; the third stage baking temperature is 85 ℃, and the baking time is 1 h.

(5) Probiotic nut packaging

Mixing semi-finished products of probiotic nuts (probiotic semen Juglandis, probiotic almond kernel, probiotic cashew kernel, probiotic hazelnut kernel) and dried fruits (cranberry, black currant raisin, and dried blueberry), packaging with nitrogen, and adding oxygen absorbent and desiccant.

Comparative example 3

(1) Preparing probiotic coating powder:

weighing 5 parts of bacillus coagulans and 350 parts of glucose powder, premixing, uniformly mixing, and putting the mixture, 350 parts of fermented yoghurt powder, 11 parts of malic acid, 16 parts of milk mineral salt, 55 parts of fructo-oligosaccharide and 3 parts of haematococcus pluvialis into a three-dimensional mixer for fully mixing;

(2) preparing mucus:

preparing coating liquid from 30 parts of maltodextrin and 90 parts of water;

(3) probiotics nut coating

Putting 10 parts of peeled walnut kernels into a coating device, and putting mucilage into a liquid storage tank at a mucilage adding end of the coating device, wherein the adding parameters of the mucilage are as follows: 0.4 part of mucus, and the time is 1min, the coating powder is put into a powder storage tank at the powder coating end, and the coating powder adding parameters are as follows: coating powder 1 part for 1min, setting the rotating speed at 40Hz, and starting the equipment. Transferring the nuts with the mucilage in the mucilage adding end to the powder coating end after 1min, simultaneously starting automatic adding of the coating powder, and transferring the coated semi-finished product to a baking tray after 1 min.

(4) Probiotic nut drying

And putting the semi-finished products in the baking tray into an oven for staged baking. The baking parameters are as follows: the first stage baking temperature is 60 ℃, and the baking time is 1 h; the second stage baking temperature is 80 ℃, and the baking time is 1 h; the third stage baking temperature is 85 ℃, and the baking time is 1 h.

(5) Probiotic nut packaging

Mixing semi-finished products of probiotic nuts (probiotic semen Juglandis, probiotic almond kernel, probiotic cashew kernel, probiotic hazelnut kernel) and dried fruits (cranberry, black currant raisin, and dried blueberry), packaging with nitrogen, and adding oxygen absorbent and desiccant.

Comparative example 4

(1) Preparing probiotic coating powder:

weighing 5 parts of bacillus coagulans, premixing with 350 parts of glucose powder to ensure that the bacillus coagulans and the glucose powder are uniformly mixed, and then putting the bacillus coagulans, the malic acid, the 16 parts of milk mineral salt, 55 parts of fructo-oligosaccharide and 3 parts of maltodextrin together into a three-dimensional mixer for fully mixing;

(2) preparing mucus:

preparing coating liquid from 5 parts of isomalt, 30 parts of maltodextrin and 90 parts of water;

(3) probiotics nut coating

Putting 10 parts of peeled walnut kernels into a coating device, and putting mucilage into a liquid storage tank at a mucilage adding end of the coating device, wherein the adding parameters of the mucilage are as follows: 0.4 part of mucus, and the time is 1min, the coating powder is put into a powder storage tank at the powder coating end, and the coating powder adding parameters are as follows: coating powder 1 part for 1min, setting the rotating speed at 40Hz, and starting the equipment. Transferring the nuts with the mucilage in the mucilage adding end to the powder coating end after 1min, simultaneously starting automatic adding of the coating powder, and transferring the coated semi-finished product to a baking tray after 1 min.

(4) Probiotic nut drying

And putting the semi-finished products in the baking tray into an oven for staged baking. The baking parameters are as follows: the first stage baking temperature is 60 ℃, and the baking time is 1 h; the second stage baking temperature is 80 ℃, and the baking time is 1 h; the third stage baking temperature is 85 ℃, and the baking time is 1 h.

(5) Probiotic nut packaging

Mixing semi-finished products of probiotic nuts (probiotic semen Juglandis, probiotic almond kernel, probiotic cashew kernel, probiotic hazelnut kernel) and dried fruits (cranberry, black currant raisin, and dried blueberry), packaging with nitrogen, and adding oxygen absorbent and desiccant.

In comparison to example 2, no BC2000 species was added in comparative example 1, no milk mineral salt was added in comparative example 2, no isomalt was added in comparative example 3, and no haematococcus pluvialis was added in comparative example 4. After eating the products of examples 2, 3 and 4 and the products of comparative examples 1 and 2 for 7 days and 28 days continuously for five groups of 50 persons, respectively, live bacillus coagulans BC2000 in feces of five groups of persons were detected (Table 6). The BC2000 is shown to germinate in the human intestinal tract, and the milk mineral salt has certain effect of promoting the proliferation of the milk mineral salt.

TABLE 6 Bacillus coagulans order of magnitude

Note: CFU/g, which means the order of magnitude of Bacillus coagulans contained in each g of feces;

BC2000 was separately cultured by passing the products of examples 2, 3, 4 and comparative examples 1, 2 through a simulated SGF sp and SIF sp, transferred to a solution to which a protein sample and a cellulose sample were added for a certain period of time, and detected by spectrophotometry. The results in Table 7 show that the strains can produce enzymes for digesting protein and cellulose, and the effect of the milk mineral salt sample is more obvious.

TABLE 7 enzymatic Activity secreted by Bacillus coagulans

Sample name	Example 2	Example 3	Example 4	Comparative example 1	Comparative example 2
						Cellulase activity U/g	96	52	170	0	49

Note: u/g, means the activity of the enzyme per g of treatment fluid;

in comparative example 4, haematococcus pluvialis, an oxygen absorbent and nitrogen are not added, the product is slightly rancid after being placed at normal temperature for 5 months, is serious after 6 months, can be smelled after being bagged after 8 months, and the product in example 1 has normal flavor and no rancid flavor after being placed at normal temperature for 8 months. Wherein the comprehensive evaluation results and the evaluation criteria of the probiotic nuts within the shelf life are shown in tables 8 and 9.

TABLE 8 comprehensive assessment of probiotic nuts on shelf life

Sample (I)	0 month	2 months old	4 months old	For 5 months	6 months old	7 months old	8 months old
								Example 1	96	94	93	92	92	90	88
Comparative example 4	96	91	85	80	75	70	65

TABLE 9 Scoring standards

Testing the storage stability of probiotic nuts: the probiotic nuts of example 1 were stored at room temperature in a dry, dark place. The detection is carried out at 0, 2, 4, 6, 7, 8 and 10 months respectively, and the survival rate of the spores is calculated. The results are shown in table 10, which indicates that the multifunctional bacillus coagulans is stable during storage of probiotic nuts.

Table 10: survival rate of BC2000 in shelf life of probiotic nuts

Sample (I)

0 month

2 months old

4 months old

6 months old

7 months old

8 months old

For 10 months

Example 1

100％

100.00％

100.00％

99.95％

99.58％

98.78％

98.42％

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.