CN114958633A - Preparation of nutrient-rich probiotic bacteria powder and application of nutrient-rich probiotic bacteria powder in goat milk powder - Google Patents
Preparation of nutrient-rich probiotic bacteria powder and application of nutrient-rich probiotic bacteria powder in goat milk powder Download PDFInfo
- Publication number
- CN114958633A CN114958633A CN202210834330.5A CN202210834330A CN114958633A CN 114958633 A CN114958633 A CN 114958633A CN 202210834330 A CN202210834330 A CN 202210834330A CN 114958633 A CN114958633 A CN 114958633A
- Authority
- CN
- China
- Prior art keywords
- nutrient
- powder
- chromium
- rich
- zinc
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 239000000843 powder Substances 0.000 title claims abstract description 114
- 235000015097 nutrients Nutrition 0.000 title claims abstract description 63
- 239000006041 probiotic Substances 0.000 title claims abstract description 55
- 235000018291 probiotics Nutrition 0.000 title claims abstract description 55
- 235000020251 goat milk Nutrition 0.000 title claims abstract description 47
- 230000000529 probiotic effect Effects 0.000 title claims abstract description 38
- 241000894006 Bacteria Species 0.000 title claims abstract description 30
- 238000002360 preparation method Methods 0.000 title claims abstract description 16
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims abstract description 83
- 239000011669 selenium Substances 0.000 claims abstract description 76
- BUGBHKTXTAQXES-UHFFFAOYSA-N Selenium Chemical compound [Se] BUGBHKTXTAQXES-UHFFFAOYSA-N 0.000 claims abstract description 73
- 229910052711 selenium Inorganic materials 0.000 claims abstract description 73
- 229940091258 selenium supplement Drugs 0.000 claims abstract description 73
- 239000011651 chromium Substances 0.000 claims abstract description 63
- 239000011701 zinc Substances 0.000 claims abstract description 63
- VYZAMTAEIAYCRO-UHFFFAOYSA-N Chromium Chemical compound [Cr] VYZAMTAEIAYCRO-UHFFFAOYSA-N 0.000 claims abstract description 56
- 229910052804 chromium Inorganic materials 0.000 claims abstract description 56
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 claims abstract description 51
- 229910052725 zinc Inorganic materials 0.000 claims abstract description 51
- 229910021556 Chromium(III) chloride Inorganic materials 0.000 claims abstract description 25
- 229960000359 chromic chloride Drugs 0.000 claims abstract description 25
- QSWDMMVNRMROPK-UHFFFAOYSA-K chromium(3+) trichloride Chemical compound [Cl-].[Cl-].[Cl-].[Cr+3] QSWDMMVNRMROPK-UHFFFAOYSA-K 0.000 claims abstract description 25
- BVTBRVFYZUCAKH-UHFFFAOYSA-L disodium selenite Chemical compound [Na+].[Na+].[O-][Se]([O-])=O BVTBRVFYZUCAKH-UHFFFAOYSA-L 0.000 claims abstract description 22
- 229960001471 sodium selenite Drugs 0.000 claims abstract description 22
- 235000015921 sodium selenite Nutrition 0.000 claims abstract description 22
- 239000011781 sodium selenite Substances 0.000 claims abstract description 22
- NWONKYPBYAMBJT-UHFFFAOYSA-L zinc sulfate Chemical compound [Zn+2].[O-]S([O-])(=O)=O NWONKYPBYAMBJT-UHFFFAOYSA-L 0.000 claims abstract description 17
- 229960001763 zinc sulfate Drugs 0.000 claims abstract description 17
- 229910000368 zinc sulfate Inorganic materials 0.000 claims abstract description 17
- 241000235070 Saccharomyces Species 0.000 claims description 35
- 230000001580 bacterial effect Effects 0.000 claims description 33
- 238000004108 freeze drying Methods 0.000 claims description 22
- 239000003223 protective agent Substances 0.000 claims description 16
- 238000000034 method Methods 0.000 claims description 14
- 238000011081 inoculation Methods 0.000 claims description 12
- 239000001963 growth medium Substances 0.000 claims description 9
- 239000010802 sludge Substances 0.000 claims description 9
- 238000012258 culturing Methods 0.000 claims description 7
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 claims description 4
- 235000020183 skimmed milk Nutrition 0.000 claims description 4
- 239000004475 Arginine Substances 0.000 claims description 2
- 239000004471 Glycine Substances 0.000 claims description 2
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 claims description 2
- HEBKCHPVOIAQTA-NGQZWQHPSA-N d-xylitol Chemical compound OC[C@H](O)C(O)[C@H](O)CO HEBKCHPVOIAQTA-NGQZWQHPSA-N 0.000 claims description 2
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 claims description 2
- 229910000397 disodium phosphate Inorganic materials 0.000 claims description 2
- 235000019800 disodium phosphate Nutrition 0.000 claims description 2
- 230000000968 intestinal effect Effects 0.000 abstract description 9
- 239000002131 composite material Substances 0.000 abstract description 4
- 239000011573 trace mineral Substances 0.000 abstract description 4
- 235000013619 trace mineral Nutrition 0.000 abstract description 4
- 238000011161 development Methods 0.000 abstract description 3
- 230000036541 health Effects 0.000 abstract description 3
- 235000013365 dairy product Nutrition 0.000 abstract description 2
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 description 79
- 239000002028 Biomass Substances 0.000 description 46
- 230000000694 effects Effects 0.000 description 45
- 210000004027 cell Anatomy 0.000 description 36
- 241001052560 Thallis Species 0.000 description 33
- 230000004044 response Effects 0.000 description 30
- 238000012360 testing method Methods 0.000 description 16
- VLZBULPKVMOFRD-UHFFFAOYSA-N [Se].[Zn].[Cr] Chemical compound [Se].[Zn].[Cr] VLZBULPKVMOFRD-UHFFFAOYSA-N 0.000 description 15
- 239000012530 fluid Substances 0.000 description 14
- 230000001965 increasing effect Effects 0.000 description 14
- 238000010586 diagram Methods 0.000 description 13
- 239000002054 inoculum Substances 0.000 description 13
- 230000002779 inactivation Effects 0.000 description 12
- 230000002829 reductive effect Effects 0.000 description 12
- 210000005253 yeast cell Anatomy 0.000 description 12
- 230000008859 change Effects 0.000 description 11
- 230000002496 gastric effect Effects 0.000 description 10
- 238000006243 chemical reaction Methods 0.000 description 9
- 102000004190 Enzymes Human genes 0.000 description 8
- 108090000790 Enzymes Proteins 0.000 description 8
- 244000005700 microbiome Species 0.000 description 8
- DQIPXGFHRRCVHY-UHFFFAOYSA-N chromium zinc Chemical compound [Cr].[Zn] DQIPXGFHRRCVHY-UHFFFAOYSA-N 0.000 description 7
- 238000000855 fermentation Methods 0.000 description 7
- 230000004151 fermentation Effects 0.000 description 7
- 239000008176 lyophilized powder Substances 0.000 description 7
- 238000005457 optimization Methods 0.000 description 7
- 239000002244 precipitate Substances 0.000 description 7
- 238000005057 refrigeration Methods 0.000 description 7
- 238000001878 scanning electron micrograph Methods 0.000 description 7
- 230000004083 survival effect Effects 0.000 description 7
- 210000004051 gastric juice Anatomy 0.000 description 6
- 238000011160 research Methods 0.000 description 6
- 239000000243 solution Substances 0.000 description 6
- 238000004458 analytical method Methods 0.000 description 5
- 210000002421 cell wall Anatomy 0.000 description 5
- 230000007423 decrease Effects 0.000 description 5
- 230000003247 decreasing effect Effects 0.000 description 5
- 238000013461 design Methods 0.000 description 5
- 238000007710 freezing Methods 0.000 description 5
- 230000008569 process Effects 0.000 description 5
- 238000003860 storage Methods 0.000 description 5
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 230000003993 interaction Effects 0.000 description 4
- 239000002609 medium Substances 0.000 description 4
- 238000001000 micrograph Methods 0.000 description 4
- 238000005070 sampling Methods 0.000 description 4
- 238000010183 spectrum analysis Methods 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- 229910052782 aluminium Inorganic materials 0.000 description 3
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 description 3
- 238000000540 analysis of variance Methods 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 238000002149 energy-dispersive X-ray emission spectroscopy Methods 0.000 description 3
- 239000011888 foil Substances 0.000 description 3
- 235000013305 food Nutrition 0.000 description 3
- 230000008014 freezing Effects 0.000 description 3
- 229910001385 heavy metal Inorganic materials 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 229910052751 metal Inorganic materials 0.000 description 3
- 239000002184 metal Substances 0.000 description 3
- 239000002245 particle Substances 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 150000003839 salts Chemical class 0.000 description 3
- 238000012216 screening Methods 0.000 description 3
- 230000001954 sterilising effect Effects 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 238000005211 surface analysis Methods 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- 231100000419 toxicity Toxicity 0.000 description 3
- 230000001988 toxicity Effects 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- 241001134770 Bifidobacterium animalis Species 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 2
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 2
- 235000013960 Lactobacillus bulgaricus Nutrition 0.000 description 2
- 241000186672 Lactobacillus delbrueckii subsp. bulgaricus Species 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 241000235342 Saccharomycetes Species 0.000 description 2
- 241000194020 Streptococcus thermophilus Species 0.000 description 2
- PTFCDOFLOPIGGS-UHFFFAOYSA-N Zinc dication Chemical compound [Zn+2] PTFCDOFLOPIGGS-UHFFFAOYSA-N 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 230000001133 acceleration Effects 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 230000004913 activation Effects 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 229940118852 bifidobacterium animalis Drugs 0.000 description 2
- 230000034303 cell budding Effects 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 235000011389 fruit/vegetable juice Nutrition 0.000 description 2
- 230000002538 fungal effect Effects 0.000 description 2
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 2
- 239000010931 gold Substances 0.000 description 2
- 229910052737 gold Inorganic materials 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 238000009616 inductively coupled plasma Methods 0.000 description 2
- MLFHJEHSLIIPHL-UHFFFAOYSA-N isoamyl acetate Chemical compound CC(C)CCOC(C)=O MLFHJEHSLIIPHL-UHFFFAOYSA-N 0.000 description 2
- 229940004208 lactobacillus bulgaricus Drugs 0.000 description 2
- 229920002521 macromolecule Polymers 0.000 description 2
- 230000004060 metabolic process Effects 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 230000003204 osmotic effect Effects 0.000 description 2
- 238000004321 preservation Methods 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 238000005507 spraying Methods 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 231100000331 toxic Toxicity 0.000 description 2
- 230000002588 toxic effect Effects 0.000 description 2
- 230000009466 transformation Effects 0.000 description 2
- 238000009777 vacuum freeze-drying Methods 0.000 description 2
- 230000037303 wrinkles Effects 0.000 description 2
- 235000013618 yogurt Nutrition 0.000 description 2
- 239000007222 ypd medium Substances 0.000 description 2
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 description 1
- 206010003210 Arteriosclerosis Diseases 0.000 description 1
- 201000001320 Atherosclerosis Diseases 0.000 description 1
- 241000186018 Bifidobacterium adolescentis Species 0.000 description 1
- 238000009631 Broth culture Methods 0.000 description 1
- 206010009944 Colon cancer Diseases 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 206010017964 Gastrointestinal infection Diseases 0.000 description 1
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 1
- 240000001046 Lactobacillus acidophilus Species 0.000 description 1
- 235000013956 Lactobacillus acidophilus Nutrition 0.000 description 1
- 241000186673 Lactobacillus delbrueckii Species 0.000 description 1
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 1
- GRYLNZFGIOXLOG-UHFFFAOYSA-N Nitric acid Chemical compound O[N+]([O-])=O GRYLNZFGIOXLOG-UHFFFAOYSA-N 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- 241000223252 Rhodotorula Species 0.000 description 1
- 241000235072 Saccharomyces bayanus Species 0.000 description 1
- ATJFFYVFTNAWJD-UHFFFAOYSA-N Tin Chemical compound [Sn] ATJFFYVFTNAWJD-UHFFFAOYSA-N 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 239000012190 activator Substances 0.000 description 1
- 239000000853 adhesive Substances 0.000 description 1
- 230000001070 adhesive effect Effects 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 208000011775 arteriosclerosis disease Diseases 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 239000003833 bile salt Substances 0.000 description 1
- 229960000074 biopharmaceutical Drugs 0.000 description 1
- 235000008429 bread Nutrition 0.000 description 1
- 238000011088 calibration curve Methods 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 1
- -1 chromium metals Chemical class 0.000 description 1
- 208000029742 colonic neoplasm Diseases 0.000 description 1
- 238000004132 cross linking Methods 0.000 description 1
- 230000001066 destructive effect Effects 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 238000004993 emission spectroscopy Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 235000020774 essential nutrients Nutrition 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000013401 experimental design Methods 0.000 description 1
- 235000021107 fermented food Nutrition 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 235000010855 food raising agent Nutrition 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 235000013402 health food Nutrition 0.000 description 1
- 230000007124 immune defense Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- 230000001788 irregular Effects 0.000 description 1
- 229940117955 isoamyl acetate Drugs 0.000 description 1
- 229940039695 lactobacillus acidophilus Drugs 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000008774 maternal effect Effects 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 150000002739 metals Chemical class 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000006872 mrs medium Substances 0.000 description 1
- 238000006396 nitration reaction Methods 0.000 description 1
- 229910017604 nitric acid Inorganic materials 0.000 description 1
- 235000006286 nutrient intake Nutrition 0.000 description 1
- 235000021231 nutrient uptake Nutrition 0.000 description 1
- 229920001542 oligosaccharide Polymers 0.000 description 1
- 150000002482 oligosaccharides Chemical class 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 230000036961 partial effect Effects 0.000 description 1
- 230000035699 permeability Effects 0.000 description 1
- 239000008055 phosphate buffer solution Substances 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 235000008476 powdered milk Nutrition 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 230000002787 reinforcement Effects 0.000 description 1
- 230000033458 reproduction Effects 0.000 description 1
- 206010039073 rheumatoid arthritis Diseases 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 235000013580 sausages Nutrition 0.000 description 1
- 238000004626 scanning electron microscopy Methods 0.000 description 1
- 229940082569 selenite Drugs 0.000 description 1
- MCAHWIHFGHIESP-UHFFFAOYSA-L selenite(2-) Chemical compound [O-][Se]([O-])=O MCAHWIHFGHIESP-UHFFFAOYSA-L 0.000 description 1
- 238000012807 shake-flask culturing Methods 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000004611 spectroscopical analysis Methods 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 239000012086 standard solution Substances 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- 208000019206 urinary tract infection Diseases 0.000 description 1
- 235000014101 wine Nutrition 0.000 description 1
- 150000003751 zinc Chemical class 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
- C12N1/16—Yeasts; Culture media therefor
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
- A23C9/00—Milk preparations; Milk powder or milk powder preparations
- A23C9/152—Milk preparations; Milk powder or milk powder preparations containing additives
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
- A23C9/00—Milk preparations; Milk powder or milk powder preparations
- A23C9/16—Agglomerating or granulating milk powder; Making instant milk powder; Products obtained thereby
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/04—Preserving or maintaining viable microorganisms
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
- C12R2001/85—Saccharomyces
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
- Y02P60/80—Food processing, e.g. use of renewable energies or variable speed drives in handling, conveying or stacking
- Y02P60/87—Re-use of by-products of food processing for fodder production
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Microbiology (AREA)
- Biomedical Technology (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Virology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Mycology (AREA)
- Botany (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses preparation of nutrient-rich probiotic bacteria powder and application of the nutrient-rich probiotic bacteria powder in goat milk powder, and relates to the technical field of nutrient-rich probiotic bacteria. According to the invention, sodium selenite, zinc sulfate and chromium trichloride are taken as a selenium source, a zinc source and a chromium source, and probiotic-Balady yeast L2 is taken as a carrier, so that the composite nutrient-rich yeast is obtained, the composite nutrient-rich probiotic goat milk powder is developed, the trace elements can be supplemented, the balance and health of intestinal flora can be promoted, and theoretical reference and technical support are provided for the preparation of two or more nutrient-rich yeast and the development of functional dairy products.
Description
Technical Field
The invention relates to the technical field of nutrient-rich probiotics, in particular to preparation of nutrient-rich probiotic bacteria powder and application of the nutrient-rich probiotic bacteria powder in goat milk powder.
Background
Selenium, zinc and chromium are essential nutrients for human and animals, play an important role in maintaining normal physiological metabolic processes, and the absorption of selenium, zinc and chromium is related to the state of selenium, zinc and chromium in free forms, the selenium, zinc and chromium are not easy to absorb, the toxicity is strong, the biological value of an inorganic state is lower than that of an organic state, and therefore, the conversion of the inorganic state of selenium, zinc and chromium into the organic state is a hotspot of research.
Probiotics, also known as probiotics or probiotics, refer to a class of beneficial microorganisms that are capable of colonizing the host by regulating the balance of the microecology, thereby promoting growth and reproduction of the host. Probiotics have been reported to have a range of therapeutic properties, such as enhancing the immune defense system, lowering serum cholesterol to prevent colon cancer, gastrointestinal and urinary infections, treating atherosclerosis, arteriosclerosis, rheumatoid arthritis, etc. Therefore, probiotics are widely used in the fields of health food, medicine, animal husbandry and the like.
Many microorganisms have the function of enriching beneficial elements, and inorganic elements can be converted into organic elements by utilizing probiotics to enrich nutrients. The human body can take in the nutrient-rich probiotics, and the effect of taking in the probiotics is realized while supplying the nutrients. The food has the health care effect of the probiotics through the fermentation of the probiotics rich in the nutrients, and can supplement other nutrients, for example, the food developed by the probiotics rich in the selenium mainly comprises fermented food such as yoghourt, wine, sausage, bread and the like or zinc-rich yoghourt leavening agent developed by the probiotics rich in the zinc and the like.
Researches by Alzate and the like find that the selenium-rich probiotics convert inorganic selenium into organic selenium, so that toxic reaction is not caused while the intake and the utilization rate of the selenium are improved. Yang et al use Streptococcus thermophilus and Lactobacillus bulgaricus to culture in a selenium-rich way under different conditions, and optimize the selenium-rich conditions by using a response surface method to obtain the optimal initial temperature, pH value and inoculum size of 40 ℃, 6.37 and 6.00 percent, 33 ℃, 5.96 and 6.73 percent respectively, wherein the enrichment rate of Streptococcus thermophilus on selenium reaches 97.05 percent, and the enrichment rate of Lactobacillus bulgaricus reaches 94.34 percent. Studies of Junqiang et al show that the selenium concentration in MRS medium is not more than 12 μ g/mL, when inoculating 5% of Bifidobacterium animalis 01 is cultured for 48h, the maximum selenium tolerance of Bifidobacterium animalis 01 is achieved, the optimal selenium concentration is 8 μ g/mL, and the effect of adding selenium and enriching selenium is optimal at 6 h. The zinc-rich mixed thallus is obtained by mixed culture of three strains of lactobacillus acidophilus ysh2, lactobacillus delbrueckii subsp bulgaricus 1.1480 and bifidobacterium adolescentis ys01 by Liudong et al.
The yeast has the characteristics of easy growth, high trace element absorption rate and the like, is an ideal carrier for enriching trace elements, but the current research mostly focuses on developing single-enriched nutrient yeast rich in selenium, zinc, iron and the like, and the research of simultaneously enriching two or more nutrients by using the yeast is not seen.
Disclosure of Invention
The invention aims to provide preparation of nutrient-rich probiotic bacteria powder and application of the nutrient-rich probiotic bacteria powder in goat milk powder, so as to solve the problems in the prior art.
In order to achieve the purpose, the invention provides the following scheme:
one of the technical schemes of the invention is as follows: the invention provides a preparation method of nutrient-rich probiotic powder, which comprises the following steps:
inoculating Balady yeast L2 into a culture medium, adding a selenium source, a zinc source and a chromium source, culturing, collecting bacterial sludge after the culture is finished, adding a freeze-drying protective agent into the bacterial sludge, and freeze-drying to obtain the nutrient-enriched probiotic bacteria powder.
Said Saccharomyces baylanicae L2 is deposited with the American type culture Collection under the accession number ATCC 9763.
Further, the selenium source, the zinc source and the chromium source are sodium selenite, zinc sulfate and chromium trichloride respectively.
Further, the concentration of the selenium source is 30 mug/mL, the concentration of the zinc source is 300 mug/mL, and the concentration of the chromium source is 300 mug/mL. The nutrient-rich probiotic powder contains 702.33-1400.91 mug/g of selenium, 890.23-4193.18 mug/g of zinc, 754.42-1700.78 mug/g of chromium and 10.75-28.20g/L of thalli.
Further, the culture conditions are as follows: the culture time is 36-60h, the inoculation amount is 5-9%, and the pH is 5.0-6.0.
Further, the culture conditions are as follows: the culture time is 48h, the inoculation amount is 8.6%, and the pH is 5.29.
Further, the freeze-drying protective agent comprises the following components in percentage by mass: 4.5% of arginine, 7% of xylo-oligosaccharide, 24% of skim milk, 0.8% of sodium hydrogen phosphate and 5.5% of glycine. The freeze-drying survival rate of the Balady yeast L2 is 83.65 percent, and the viable count of unit bacterial powder can reach 4.37 multiplied by 10 10 CFU/g。
Further, adding a freeze-drying protective agent according to the mass ratio of the bacterial sludge to the freeze-drying protective agent of 1: 1.
The second technical scheme of the invention is as follows: the invention provides the nutrient-rich probiotic powder prepared by the preparation method.
The relation of logarithm (Lgk) of the inactivation rate constant of the nutrient-rich probiotic bacteria powder to the absolute temperature (1/T) meets the equation Lgk of-5585.4/T +16.582, and the inactivation rate constants of the nutrient-rich probiotic bacteria powder under the conditions of 4 ℃ (refrigeration temperature) and-18 ℃ (freezing storage temperature) are k 4 ＝2.62×10 -5 ，k -18 ＝4.77×10 -6 。
The third technical scheme of the invention is as follows: the invention provides application of the nutrient-rich probiotic powder in goat milk powder.
The fourth technical scheme of the invention is as follows: the invention provides nutrient-rich probiotic goat milk powder which contains the nutrient-rich probiotic powder.
Further, the addition amount of the nutrient-rich probiotic powder is 0.05% -1%.
The number of the live probiotics in per gram of the nutrient-rich probiotic goat milk powder is 2.18 multiplied by 10 7 ～4.37×10 8 CFU/g。
The invention discloses the following technical effects:
according to the invention, sodium selenite, zinc sulfate and chromium trichloride are taken as a selenium source, a zinc source and a chromium source, and probiotic-Balady yeast L2 is taken as a carrier, so that the composite nutrient-rich yeast is obtained, the composite nutrient-rich probiotic goat milk powder is developed, the trace elements can be supplemented, the balance and health of intestinal flora can be promoted, and theoretical reference and technical support are provided for the preparation of two or more nutrient-rich yeast and the development of functional dairy products.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.
FIG. 1 is a scanning electron micrograph of Saccharomyces baylanicae L2, a and b are scanning electron micrographs at 5K and 10K, respectively;
FIG. 2 is a graph of the effect of sodium selenite concentration on selenium content and biomass of Saccharomyces baylanicae L2;
in FIG. 3, a-j are scanning electron micrographs of L2 cells of Baladia yeast cultured at different sodium selenite concentrations, wherein a, c, e, g and i are magnified by 5K, and b, d, f, h and j are magnified by 10K;
FIG. 4 is a graph of the effect of zinc sulfate concentration on zinc content and biomass of Saccharomyces baylanicae L2;
in FIG. 5, a-j are scanning electron micrographs of L2 cells of Baladia yeast cultured at different zinc sulfate concentrations, wherein a, c, e, g and i are magnified by 5K, and b, d, f, h and j are magnified by 10K;
FIG. 6 is a graph of the effect of chromium trichloride concentration on chromium content and biomass of Saccharomyces baylanicae L2;
FIG. 7 is a scanning electron micrograph of L2 cells of Saccharomyces baylanica cultured at a-j different concentrations of chromium trichloride, wherein a, c, e, g and i are magnified 5K, and b, d, f, h and j are magnified 10K;
FIGS. 8(a) - (d) are the effect of different salt concentrations and nutrient combinations on enrichment and growth of Saccharomyces baylanicae L2, respectively;
FIG. 9 is a graph of the effect of culture conditions on Saccharomyces baylanicae L2 for the production of selenium-enriched zinc-chromium Saccharomyces baylanicae L2, wherein (a) is the culture time; (b) is pH; (c) the inoculation amount is shown;
FIG. 10 is a graph showing the effect of culture time, pH and inoculum size of Saccharomyces baylanicae L2 on selenium content: (a) the (b) and the (c) are 2D contour diagrams, and the (D) and the (e) and the (f) are 3D response surface diagrams;
FIG. 11 shows the effect of incubation time, pH and inoculum size of Saccharomyces baylanicae L2 on zinc content: (a) the (b), (c) are 2D contour diagrams, and the (D), (e) and (f) are 3D response surface diagrams;
FIG. 12 is a graph showing the effect of culture time, pH and inoculation amount of Saccharomyces baylanicae L2 on chromium content: (a) the (b) and the (c) are 2D contour diagrams, and the (D) and the (e) and the (f) are 3D response surface diagrams;
FIG. 13 shows the effect of cultivation time, pH and inoculum size on the biomass of Saccharomyces baylanicae L2: (a) the (b) and the (c) are 2D contour diagrams, and the (D) and the (e) and the (f) are 3D response surface diagrams;
FIG. 14 is a scanning electron micrograph of selenium zinc chromium enriched Saccharomyces baylasiae L2; wherein a and b are scanning electron micrographs with the magnification of 5K and 10K respectively;
FIG. 15 is an EDX analysis spectrum of selenium zinc chromium enriched Saccharomyces baylasiae L2;
FIG. 16 is a graph of the effect of different lyoprotectants on the yield (a) of Bayland yeast L2 bacterial powder, the freeze-drying survival rate and the viable count (b) per unit of bacterial powder;
FIG. 17 is a test of the acceleration of freeze-dried powder of selenium-zinc-chromium-enriched Saccharomyces bailadi L2; wherein (a): the change of viable count with time; (b) the method comprises the following steps arrhenius diagram;
FIG. 18 is an accelerated test of lyophilized bacteria powder in goat milk powder; wherein (a): the change of viable count with time; (b) the method comprises the following steps arrhenius diagram;
fig. 19 shows the viable count change of probiotic goat milk powder after being treated in simulated gastric fluid (a) and intestinal fluid (b) for different time.
Detailed Description
Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.
As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including, but not limited to.
The Baylandi yeast L2 used in the examples of the present invention was deposited as lyophilized powder in laboratory 1C419 of the institute of food and bioengineering, university of Shaanxi science and technology, which was derived from American type culture Collection strain ATCC 9763.
The main experimental reagents used in the examples of the present invention are shown in table 1:
TABLE 1
Example 1
1. Activation of bacterial strains
Dissolving lyophilized powder of Balady yeast L2 in YPD medium, culturing in shaking incubator at 30 deg.C and 180r/min for 24 hr, and continuously activating for three generations with inoculation amount of 5% (v/v) to make viable count of each strain reach 10 8 CFU/g, which was kept in a refrigerator at 4 ℃ until use.
2. Observation of Saccharomyces bayanensis L2 by electron microscope
Under the aseptic condition, inoculating 5% (v/v) of activated Balady yeast L2 into a YPD broth culture medium, placing the culture medium in an incubator at 30 ℃, shaking and culturing for 24h at 180r/min, after the growth of the strain reaches a logarithmic phase, centrifugally collecting thalli, and measuring the shape of the thalli by using SEM (scanning electron microscope), as shown in figure 1, the Balady yeast L2 is complete, ellipsoidal, large in shape and volume, and a small number of round cells are arranged, and buds appear around part of maternal cells, which indicates that the Balady yeast L2 has the common characteristics of yeasts and budding. In FIG. 1, a is 5kx and b is 10 kx.
3. Determination of nutrient conversion
And (3) determination of conversion rate: and detecting the contents of the sodium selenite, the zinc sulfate and the chromium trichloride by using inductively coupled plasma emission spectroscopy (ICP-AES). Centrifuging the yeast transformation liquid rich in nutrients, taking 5mL of supernatant, adding nitric acid and hydrogen peroxide for nitration, diluting the nitrated sample to 20mL by pure water, placing the sample in ICP-AES for detecting the contents of selenium, zinc and chromium, and calculating the contents according to a calibration curve of a corresponding standard solution established in ICP.
Conversion (%) [ (B-a)/B ] × 100 (1)
In the formula:
a, detecting the content of the obtained selenium, zinc and chromium;
b-total content of selenium, zinc and chromium added.
Yeast biomass determination: and (3) after shake-flask culture, centrifuging the fermentation liquor at 6000r/min for 10min, collecting thalli, washing the thalli for 3 times by using distilled water, collecting yeast, and drying the yeast at 105 ℃ to constant weight to obtain the yeast biomass.
4. Scanning electron microscope and energy spectrum analysis (SEM + EDX)
The nutrient-rich yeast transformation solution was centrifuged to remove the supernatant, and 2.5% glutaraldehyde was added to the sludge and fixed for 3 hours. Washing the precipitate with phosphate buffer solution (pH7) for 10min for 2 times after centrifugation, continuously and gradiently eluting the washed precipitate with ethanol (30%, 50%, 70%, 80%, 90%, 100%) for 10min for each time, finally washing with isoamyl acetate, and vacuum freeze-drying the precipitate for 30h to obtain the yeast powder rich in nutrients. Taking a proper amount of nutrient-rich yeast powder, placing the nutrient-rich yeast powder on a sample carrying table containing metal conductive adhesive, carrying out gold spraying treatment, placing a sample subjected to gold spraying in a sample chamber of an electron microscope for observation, wherein the acceleration voltage is 2KV, and the magnification is 10000 times. The cell surface precipitates observed in the scanning electron microscope were qualitatively analyzed for elements and further analyzed with an X-ray energy spectrometer.
a. Influence of sodium selenite concentration on enrichment effect of Balady yeast L2 and scanning electron microscope image analysis
The sodium selenite concentration was set to 10, 20, 30, 40 and 50 μ g/mL, and the selenium content and biomass in yeast were measured and observed by scanning electron microscopy, and the results are shown in fig. 2 and 3. In FIG. 3, a and b are 10. mu.g/mL; c, d is 20 mu g/mL; e, f is 30 mu g/mL; g, h is 40 mu g/mL; i, j is 50. mu.g/mL, a, c, e, g, i is 5kx, b, d, f, h, j is 10 kx.
As can be seen from FIG. 2, when the concentration of sodium selenite is less than 30. mu.g/mL, the selenium content and biomass of the Saccharomyces bayanensis L2 enriched in the cells increased with the increase of the concentration of sodium selenite; when the concentration reaches 30 mug/mL, the selenium content and the biomass of the thalli reach maximum values, namely 1060.59 mug/g and 27.2g/L respectively; when the concentration of sodium selenite is more than 30 mug/mL, the growth of the thalli is inhibited, so that the selenium-rich amount and the biomass of the thalli are gradually reduced, mainly because the reduction of selenite is mainly performed by related reductase in the thalli, when the sodium selenite is excessive, the thalli are saturated in the conversion of the sodium selenite, and the activity of the thalli is inhibited by the sodium selenite, so that the action activity of related enzyme is reduced.
The selenium concentration in the culture medium can influence the growth of thalli and the enrichment of selenium, so that the inhibition effect of selenium on the growth of thalli is reduced in the yeast fermentation process, the maximum growth of the thalli is ensured, and the conversion of the thalli to the selenium is improved to obtain high-content organic selenium protein. Observing the figure 3, when the concentration of the sodium selenite is 10-30 mug/mL, the wrinkles on the cell surface gradually disappear and fine cracks appear compared with the control group. When the concentration is more than 30 mug/mL, few cells keep a normal state, and most cells are sunken and deformed. In general, the sodium selenite concentration was chosen to be 30. mu.g/mL.
b. Influence of zinc sulfate concentration on enrichment effect of Balady yeast L2 and scanning electron microscope image analysis
The zinc sulfate concentrations were set at 200, 300, 400, 500 and 600. mu.g/mL for a conversion time of 24h, and the results are shown in FIGS. 4 and 5. In FIG. 5, a and b are 200. mu.g/mL; c, d is 300 mu g/mL; e, f is 400 mu g/mL; g, h is 500 mu g/mL; 600. mu.g/mL of i, j, 5kx of a, c, e, g and i, and 10kx of b, d, f, h and j.
As can be seen from FIG. 4, the zinc content and biomass of the enriched cells of the Saccharomyces baylanicae L2 both decreased with the increase of the concentration of zinc sulfate, and the maximum values of zinc content and biomass were reached at a concentration of 300. mu.g/mL, respectively, 3164.76. mu.g/g and 35.45 g/L. When the concentration reached 600. mu.g/mL, the zinc content and the biomass were reduced to 1375.2. mu.g/g and 15.55g/L, respectively. The zinc ion is a cofactor of a plurality of biological enzymes in organisms, and promotes the growth and development of the yeast along with the increase of the concentration of zinc sulfate. However, the yeast can tolerate a limited zinc concentration and osmotic pressure, and when the zinc salt is excessive, the enzyme activity of the yeast cells is not activated, and the growth of the yeast cells is limited instead by increasing the zinc ion concentration to a certain degree, so that the yeast cells are poisoned and poisoned, and the enzyme activity of the yeast cells is inhibited.
As can be seen from FIG. 5, Zn was contained in the medium 2+ When the concentration of (A) is 200 mug/mL, the cell morphology is mainly round or budding, and the volume is slightly reduced; when the concentration is increased to 300 mu g/mL, the cell shape is mainly circular, the wrinkles on the cell surface are reduced, the cell surface is smoother and smoother, and precipitates begin to appear; when Zn is present in the medium 2+ When the concentration reaches 500 mug/mL, the morphology of a few cells is normal, the morphology of most yeast cells is irregular, and the cells are sunken.
Zn in yeast cells 2+ The main site of enrichment is the cell wall, during the process of synthesizing the relevant enzymes by the cell wall of yeast cell, Zn 2+ Can be used as activator and inhibitor, and low concentration Zn is added 2+ Related enzymes can be activated to increase the biomass of the thalli; and high concentration of Zn 2+ Not only the osmotic pressure of the culture solution is influenced, but also the activity of partial enzyme is inhibited, and the synthesis of yeast cell walls is influenced, thereby influencing the biomass and the shape of the yeast. Therefore, the zinc sulfate is finally determined by combining the zinc content, the biomass and the scanning electron microscope resultThe concentration was 300. mu.g/mL.
c. Influence of chromium trichloride concentration on enrichment effect of Saccharomyces baylanicae L2 and scanning electron microscope image analysis
The results of culturing Saccharomyces bayanensis L2 in YPD medium with chromium trichloride concentrations of 100, 200, 300, 400 and 500. mu.g/mL, and measuring chromium content and biomass in the cells are shown in FIGS. 6 and 7. In FIG. 7, a and b are 100. mu.g/mL; c, d is 200 mug/mL; e, f is 300 mu g/mL; g, h is 400 mu g/mL; i, j is 500. mu.g/mL, a, c, e, g, i is 5kx, b, d, f, h, j is 10 kx.
FIG. 6 shows that the biomass of Saccharomyces baylanicae L2 varied in media with different concentrations of chromium trichloride. When the concentration of the chromium trichloride in the culture medium is 100-300 mu g/mL, the biomass also slowly increases along with the increase of the concentration; when the concentration of the chromium trichloride is 300 mu g/mL, the biomass reaches the maximum value of 27.725 g/L; when the concentration of chromium trichloride in the medium was higher than 300. mu.g/mL, the biomass decreased rapidly with increasing concentration. The maximum biomass of P.parapsilosis L2 was not present in the medium with the lowest concentration of chromium trichloride but in a concentration of 300. mu.g/mL. The growth of yeast is stimulated mainly by low concentration of chromium, while the growth and metabolism of yeast is inhibited by the toxic action of chromium element when the concentration of chromium is too high. When the chromium content of the thalli is high, the thalli amount is very low; conversely, when the amount of bacteria is higher, the chromium content is lower.
From FIG. 7, it can be seen that the enriched precipitate on the yeast cell wall gradually increased with the increase of the chromium trichloride concentration, as compared with the Saccharomyces baylanicae L2 (FIG. 1) cultured without addition of chromium trichloride. At chromium trichloride concentrations of 100. mu.g/mL and 200. mu.g/mL, no change in yeast cell shape occurred. The yeast cell shape becomes longer with concentrations of 300. mu.g/mL and 400. mu.g/mL. When the concentration reached 500. mu.g/mL, the cell deformation was increased and the surface was even dented. However, the cell walls of Saccharomyces baylanicae L2 were not significantly disrupted at each chromium trichloride concentration in the figure, indicating that they can normally reproduce at the above-mentioned chromium trichloride concentrations.
The microorganisms exhibit certain adaptability and resistance to harmful substances and environmental changes. Chromium as heavy metal has certain toxicity to microorganism. As can be seen from the scanning electron microscope image of the chromium-rich yeast, the yeast cell wall begins to precipitate along with the increase of the concentration of chromium trichloride (100-500 mug/mL). The yeast secretes biological macromolecules such as protein, lipid and the like to the outside of the body in the process of enriching chromium, and the biological macromolecules are combined with the chromium to form particles to be attached to the surface of cells; or the chromium concentration on the surface of the thallus is too high so as to form certain destructive effect. leaf-Yangshao et al studied the adsorption of heavy chromium metals by yeast and found that certain heavy metals, at low concentrations, promote the growth of microorganisms, mainly because these metals are essential components of certain enzymes in the microbial cells; however, when the heavy metal content exceeds its critical concentration, it has a certain toxicity to microorganisms and can even kill microorganisms. Researches by Zhang Xiaoqing and the like find that the structure of the saccharomycetes before adsorbing the lead is oval and regular in shape; the surface of the yeast after adsorbing the lead is rough and uneven. This indicates that the cell surface material is associated with Pb 2+ The effect is that the resulting precipitate adheres to the cell wall, causing a change in cell morphology. Cr in the invention 3+ Action on Saccharomyces baylanicae L2 with Pb 2+ Similar effects on the cell surface. According to the comprehensive measurement of chromium content, biomass and scanning electron microscope result analysis, the concentration of chromium trichloride in the selected culture medium is 300 mug/mL.
5. Different nutrient concentration combinations and selenium, zinc and chromium enrichment amount
The enrichment conditions of thalli under different salts and different concentration combinations are researched by taking three salt concentrations obtained by a single-factor test as the center. Sodium selenite, zinc sulfate and chromium trichloride with different concentrations are added into a culture medium and are added in 4 groups of different combinations, which are respectively: group A Se + Zn; group B Se + Cr; group C Zn + Cr; group D Se + Zn + Cr. In the group A, the concentration 1 is 10+100 mug/mL, the concentration 2 is 15+150 mug/mL, and the concentration 3 is 30+300 mug/mL; in the group B, the concentration 1 is 10+100 mug/mL, the concentration 2 is 15+150 mug/mL, and the concentration 3 is 30+300 mug/mL; in the group C, the concentration 1 is 100+100 mug/mL, the concentration 2 is 150+150 mug/mL, and the concentration 3 is 300+300 mug/mL; in group D, the concentration 1 was 10+100+ 100. mu.g/mL, the concentration 2 was 15+150+ 150. mu.g/mL, and the concentration 3 was 30+300+ 300. mu.g/mL. And measuring the amount of selenium, zinc and chromium enriched in the yeast and the biomass after the culture is finished.
The effect of different nutrient combinations and concentrations on enrichment and growth of Saccharomyces baylanicae L2 was studied, and the results are shown in FIG. 8, (a): se + Zn; (b) the method comprises the following steps Se + Cr; (c) the method comprises the following steps Zn + Cr; (d) the method comprises the following steps Se + Zn + Cr.
As can be seen from FIG. 8, the enrichment amounts of selenium, zinc and chromium in the yeasts in group 4 of experiments all increased with the increase of the concentration, and the biomass of the cells decreased to different degrees. In the group A, the selenium content and the zinc content in the yeast are gradually increased along with the increase of the concentrations of the sodium selenite and the zinc sulfate, the selenium content and the zinc content of unit thalli reach 732.5 mug/g and 1407.77 mug/g at the concentration of 3, and the biomass reaches 26.78 g/L. In group B, the selenium content and chromium content per cell at concentration 3 reached 533.745 μ g/g and 632.02 μ g/g. The biomass change was evident in group C. The group D is enriched with three nutrients simultaneously, and at the concentration of 3, the selenium content, the zinc content and the chromium content reach 588.76 mug/g, 1061.54 mug/g and 930.57 mug/g, and the biomass is 21.75 g/L. Comprehensively considering the yield of the yeast and the effect of enriching selenium, zinc and chromium, the test results in group D (30 mug/mL of sodium selenite, 300 mug/mL of zinc sulfate and 300 mug/mL of chromium trichloride) are selected for the next research under the test conditions.
6. Design of optimized response surface of selenium-rich zinc-chromium yeast culture condition
According to the early screening result, a strain with strong capability of enriching three nutrients is obtained, in order to further improve the enrichment capability of probiotics on the three nutrients, a single-factor test is carried out on the strain, the influence of the culture time (24-72h), the inoculation amount (3-11%), and the pH (4.5-6.5) on the enriched nutrients of the saccharomycetes is researched, the most significant influence factor and the optimal value thereof are selected as central points according to the single-factor result, the design of a 3-factor 3 horizontal response surface is carried out, the process for preparing the selenium-zinc-chromium-enriched yeast is optimized, and the optimal process condition is obtained and verified.
(1) Effect of culture conditions on the preparation of selenium-Zinc-chromium-enriched Saccharomyces bayanensis L2
In order to explore the influence of the culture conditions on the enrichment effect and growth of the Baylandi yeast L2, single-factor tests are carried out on the culture time, the initial pH value and the inoculum size by taking 5 percent of the inoculum size, the culture temperature of 30 ℃ and the shaking table rotating speed of 180r/min as basic culture conditions, the enrichment amount of three nutrients and the biomass of thalli are measured, the influence is examined, and the result is shown in figure 9. In FIG. 9, (a) - (c) are the effects of culture time, initial pH and inoculum size on the enrichment of three nutrients and biomass, respectively.
As can be seen from FIG. 9(a), the biomass and the enrichment amount of yeast tended to increase and decrease with time. At 48h, the biomass and the selenium content of the thalli respectively reach the maximum values of 27.325g/L and 1135.685 mu g/g. At 36h, the zinc-rich amount and the chromium-rich amount of the bacterial cells reach maximum values of 3044 mu g/g and 1346.25 mu g/g respectively. Two aspects of consideration are combined. The culture time of the Balady yeast L2 is preferably 48 hours.
As can be seen from FIG. 9(b), the biomass and enrichment of the biomass of the cells both increased and then decreased with increasing pH. At pH 5.5, the selenium content and chromium content of the thallus reach maximum values of 1280.851 mug/g and 1159.609 mug/g respectively. At this point, the biomass was 27.99 g/L. At pH 6, the zinc content of the enriched cells reached a maximum of 3990.493. mu.g/g. When the pH value continues to increase, the content of the enriched nutrients of the thalli is reduced, and the biomass is also reduced. Suhajda et al evaluated the effect of culture conditions on selenium uptake by Saccharomyces cerevisiae, the most important effects being acidity and dissolved oxygen content in the culture medium. And comprehensively considering the pH value of 5.5 to carry out the next process optimization.
The synthesis of useful biologicals by microorganisms depends on nutrient consumption, which in turn depends to a large extent on the density of the cells in a limited volume of culture. This requires control of the inoculum size of the cells to ensure optimal nutrient uptake for high product synthesis. In fig. 9(c), different inoculum sizes had different effects on yeast biomass, selenium, zinc and chromium enrichment. When the inoculation amount is 7%, the selenium content, the zinc content and the biomass of the thalli reach the maximum values of 25.935g/L, 1302.74 mu g/g and 3686.83 mu g/g. At an inoculum size of 9%, the chromium content reached a maximum of 1260.224. mu.g/g. The influence of yeast biomass and thalli on two factors of the enrichment amount of each nutrient is comprehensively considered, and then 7 percent of Balady yeast L2 is selected for further optimization.
(2) Response surface method optimization of selenium-rich zinc-chromium Bailadi yeast L2 fermentation process
Based on the above results of the one-factor test, a three-factor three-level Box-Behnken test design of N-15 was performed for a (cultivation time), b (ph), and C (inoculum size) with the response values of rhodotorula parapsilosis L2R1 (selenium content), R2 (zinc content), R3 (chromium content), and R4 (biomass). The coding tables for the factor levels are shown in Table 2, and the experimental design and results are shown in Table 3.
TABLE 2 factor level table for Box-Behnken test optimization of fermentation process of Bailadi yeast L2 rich in selenium, zinc and chromium
TABLE 3 Box-Behnken test design and results for selenium-zinc-chromium-enriched Bailadi yeast L2 fermentation process optimization
From the test results in the table above, a regression equation was constructed as follows:
R1＝1242.06-134.07A+1.22B-19.32C+148.95AB-120.80AC-40.35BC-300.38A 2 +85.94B 2 +27.49C 2
R2＝2830.96-466.66A-176.47B+124.88C+83.65AB-965.88AC-175.80BC-912.03A 2 -588.53B 2 +511.85C 2
R3＝1535.86-87.75A-56.84B+34.48C+18.40AB-69.28AC-191.48BC-583.38A 2 -89.01B 2 -73.12C 2
R4＝26.67-1.36A+0.19B-0.32C+2.29AB-2.69AC+0.44BC-8.10A 2 -2.41B 2 -1.02C 2
analysis of variance (ANOVA) can be used to assess the significance of the coefficients in the model,f values to determine the effect of different variables. The p-value indicates the degree of influence of the factor on the results and is lower the more pronounced. R 2 The correlation (predicted value and model) was represented, and the analysis of variance results are shown in table 4.
Table 4 response surface analysis of variance table
Note: indicates extreme significance (p <0.001), 'indicates very significance (p <0.01), and' indicates significance (p <0.05)
From table 4, it can be seen that the response values R1 (selenium content), R2 (zinc content), R3 (chromium content) and R4 (biomass) regression models are all p values less than 0.05, and the mismatching terms are all greater than 0.05, indicating that the models are very significant and the mismatching terms are not significant, and the models are usable. For the response value R1 (selenium content), the equation determines the coefficient (R) 2 ) The value of (c) is 99.58%, indicating that the equation can account for response changes in excess of 99.58%. Adjusting the coefficient of determination (R) 2 Adj ) The value of (a) is 98.83% and the difference from the coefficient of determination is only 0.65%, which further indicates that the regression equation and the experimental data fit well. First order A, second order A 2 And the p-value of the interaction term AB is less than 0.01, indicating that they contribute more to the model.
Equation determining coefficient (R) of response value R2 (zinc content) 2 ) The value was 95.76%, indicating that the analysis can be used to interpret the 95.76% experimental results. R adj 2 88.12%, and R 2 More recently, the model has better fitting performance. First order term A and second order term A 2 The p-value of the interaction term AC is less than 0.01, indicating that it contributes more to the model.
Determination of the response value R3 (chromium content) coefficient R 2 ＝98.70％，R adj 2 96.37%, indicating that the regression equation fits well to the experimental data. And A is 2 P-values of less than 0.001 indicate a significant effect on the model.
Correlation coefficient (R) of response value R4 (biomass) 2 0.9084) was closer to 1, indicating that the model for predicting L2 biomass of P.parapsilosis isReliable, regression equations may be used to predict the response value R4 instead of experimental points of truth.
Response surface analysis was performed on the regression model using Design-expert software to obtain a 2D contour map and a 3D response surface map of the response value R1 (selenium content) in fig. 10. As can be seen from fig. 10, there is interaction between AB, AC and BC, and each three-dimensional surface map is substantially arched, i.e., the response value R1 appears to rise to fall with the change of each factor, and has a maximum value.
Contour plots and response plots of the effect of incubation time, pH and inoculum size on response R2 (zinc content) are shown in FIG. 11.
As can be seen from fig. 11, the contour diagrams are all elliptical, and the 3D diagrams are all arched with protrusions, which indicates that when the concentration of one of the three substances is fixed, the viable count increases with the concentration of the other two substances, the viable count tends to increase first and then decrease, and the maximum value is near the center point.
The regression model was subjected to response surface analysis, and a contour map and a response surface map of each factor with respect to the response value R3 (chromium content) were obtained, with the results shown in fig. 12.
The contour plots in FIG. 12 are all approximately elliptical, with three 3D response surface plots approximately dome-shaped with the opening facing downward, illustrating that the chromium content per cell increases first and then decreases as time and inoculum size of the cell increases when pH is constant; when the inoculation amount of the bacteria is not changed, the chromium content of the unit bacteria is increased and then reduced along with the increase of the pH value and the culture time; when the culture time is not changed, the chromium content of the unit thalli is increased and then reduced along with the increase of the inoculation amount and the pH value of the thalli. And maxima occur near the center point.
The experimental residuals of the response R4 (biomass) and the experimental true versus predicted values are shown in fig. 13. From fig. 13, it can be seen that there are interactions between the factors and that a model is available.
After the regression equation is analyzed by using Design-expert 8.0.6 software, a predicted value of the optimal process parameter for preparing the selenium-zinc-chromium-enriched Saccharomyces bailadi L2 is obtained. The predicted optimal process conditions comprise 48h of culture time, 5.29 of pH value and 8.6 percent of inoculation amount, and under the conditions, the selenium content in a unit thallus is 1416.39 mu g/g, the zinc content is 4402.558 mu g/g, the chromium content is 1435.859 mu g/g and the thallus biomass is 18.527 g/L. Three groups of parallel tests prove that the biomass of the thalli is 17.86g/L, the selenium content in unit thalli is 1405.01 mug/g, the zinc content is 4202.558 mug/g, and the chromium content is 1398.77 mug/g, which are respectively improved by 1.38, 2.95 and 0.5 times compared with a control group (the selenium content is 588.76 mug/g, the zinc content is 1061.54 mug/g and the chromium content is 930.57 mug/g). The conversion rates of the organic zinc and the organic chromium respectively reach 89.44%, 94.28% and 87.15%. The actual test value is very close to the predicted model value, which shows that the optimization of the process for preparing the selenium-zinc-chromium-enriched Bailaddick yeast L2 is feasible by adopting the Box-Behnken response surface design.
7. Microscopic morphology observation and energy spectrum analysis of selenium-rich zinc-chromium Bailandi yeast L2
Scanning electron microscope observation and X-ray energy spectrum analysis were performed on selenium-zinc-chromium-enriched saccharomyces bayanus L2 obtained by response surface optimization, and the results are shown in fig. 14 and 15. In FIG. 14, a is 5kx and b is 10 kx.
Compared with the control group shown in figure 1, the surfaces of the thalli which are rich in various nutrients are invaginated and shrunk, and the surfaces of the thalli are provided with round particles. Compared with the fig. 3, fig. 5 and fig. 7, the thallus is changed from singly enriching one element to simultaneously enriching three elements, the change of the cell morphology is more obvious, and the particulate matters on the surface of the thallus are also obviously increased. EDX spectroscopy was performed on these particles to obtain the spectral analysis chart of fig. 15. From the figure, it can be seen that there are a characteristic peak of selenium at 1.204KeV, a characteristic peak of zinc at 2.123KeV, and a characteristic peak of chromium at 0.573 KeV. The Balady yeast L2 is explained to biologically convert sodium selenite, zinc sulfate and chromium trichloride to obtain organic selenium, zinc and chromium elements which are distributed on the surface of the thallus and outside the thallus.
Example 2 preparation of lyophilized powder of selenium-zinc-chromium-enriched Saccharomyces bayanensis L2
Inoculating the seed fermentation liquid activated to 3 generations into YPD according to the inoculation amount of 5%, simultaneously adding sodium selenite, zinc sulfate and chromium trichloride, culturing at 37 deg.C for 24h, and centrifuging to collect bacterial sludge (4 deg.C, 8000r/min, 20 min). Adding the freeze-drying protective agent according to the ratio of the bacterial sludge to the freeze-drying protective agent of 1:1, carrying out freeze-drying by using a vacuum freeze-drying machine, and screening the freeze-drying protective agent on the basis of laboratory preliminary study, wherein the screening is shown in table 5.
Preparing a protective agent solution: dissolving one or more of saccharide, macromolecular protectant and polymer protectant in distilled water at certain concentration, stirring, sterilizing at 121 deg.C for 15min, and storing in 4 deg.C refrigerator; sterilizing skim milk at 95 deg.C for 30min, and filtering amino acids with 0.22 μm filter membrane for sterilization.
TABLE 5 Freeze-drying protectant species
FIG. 16 is a graph showing the effect of different lyoprotectants on the yield of Bayland yeast L2 fungal powder (panel a), the freeze-drying survival rate, and the viable count per unit of fungal powder (panel b). (C for control, C1, C2, C3, C4, C5 are 5 kinds of lyoprotectants) (. about.P.0.05,. about.P.0.01,. about.P.0.001). As can be seen in FIG. 16(a), the presence or absence of the lyoprotectant resulted in a large difference in the amount of lyophilized powder of Saccharomyces baylanicae L2. The yield difference of the bacterial powder of other 4 groups is very significant (P <0.001) by taking the control group C as a reference, which indicates that the addition of the freeze-drying protective agent has a significant effect on the bacterial powder yield of the freeze-dried Baylandi yeast L2. The freeze-dried powder yield of the control group is minimum because the anti-freezing factor and the freeze-drying protective agent are not added in the control group. The C1 bacterial powder is only added with 30% of skim milk and does not generate cross-linking effect with other liquid, under the vacuum effect, a part of solid matters are scattered in the cavity of the dryer, and the yield of the bacterial powder is relatively low. The highest amount (10.29g/L) of the bacterial powder C4 is added to the bacterial powder C2, C3, C4 and C5 in the formula of the protective agent, so that the yield of the bacterial powder is improved.
As can be seen from FIG. 16(b), the freeze-drying survival rate of the bacterial powder of the other 4 groups was very significant (P) with reference to the control group C<0.001). The freeze-drying survival rate of the C4 bacterial powder is the highest, which reaches 83.65%, the viable count of the unit bacterial powder can reach 10.64log CFU/g, namely the viable count is 4.37 multiplied by 10 10 CFU/g shows that the formula effect of the freeze-drying protective agent determined by preparing the bacterial powder of the No. C4 strain is obvious, and the freeze-drying protective agent is an ideal anti-freezing protective agent prepared from the freeze-drying bacterial powder of the Bayladdi yeast L2.
Example 3 storage stability study of selenium-zinc-chromium-enriched Saccharomyces baylanica L2 powder
(1) The prepared freeze-dried bacterial powder samples are respectively packaged in small aluminum foil bags, the freeze-dried bacterial powder is packaged in tin foil bags, and the small aluminum foil bags are respectively placed in water baths at 45 ℃, 50 ℃ and 55 ℃. Taking the viable count as an evaluation index, sampling and measuring the viable count once every 2 hours until 12 hours.
(2) Adding the Baylandi yeast L2 into the goat milk powder according to the adding amount of 1% by mass, uniformly mixing by using a three-dimensional mixer, subpackaging in aluminum foil bags, and evaluating the storage stability of the Baylandi yeast L2 in the goat milk powder by measuring the viable count at different accelerated temperatures. Sampling at 45 deg.C, 50 deg.C and 55 deg.C every 2h to determine viable count of once goat milk powder, up to 12 h.
The inactivation rate constants k of the selenium-rich zinc-chromium Bailadi yeast L2 and the goat milk powder thereof at different temperatures are calculated by the formula 2, and the inactivation rate constants k of the selenium-rich zinc-chromium Bailadi yeast L2 and the goat milk powder thereof at different stabilities are calculated according to the formula 3 to predict the stabilities of the bacteria and the goat milk powder thereof.
lgN t -lgN 0 ＝kt (2)
In the formula N 0 Is the initial viable count of the sample, N t The number of viable bacteria at the moment t of the sample is CFU/mL; k and k 0 Is a rate constant in units of h -1 ；E a Is activation energy, in units of J/mol; r is an ideal gas constant with the unit of J/mol.K; t is the absolute temperature in K.
a. Study on storage stability of selenium-rich zinc-chromium Bailadi yeast L2 bacterial powder
The best protectant formula of the selenium-zinc-chromium-enriched Bailadi yeast L2 is used for preparing the freeze-dried fungus powder. According to the above experimental method, it is estimated that the number of viable bacteria of the freeze-dried powder of Bayland yeast is 1 year when the powder is preserved at 4 ℃ (refrigeration temperature) and-18 ℃ (freezing temperature). The results are shown in FIG. 17.
As can be seen from FIG. 17(a), Lg (Nt/N0) decreases with time, i.e., DunalideThe survival rate of yeast L2 decreased. From the graph, the inactivation rate constants of the freeze-dried powder of the saccharomyces bayanensis L2 at different temperatures are respectively as follows: k is a radical of 45 ＝0.1048(R 2 ＝0.9901)、k 50 ＝0.1922(R 2 ＝0.9960)、k 55 ＝0.3597(R 2 0.9898). The inactivation rate constants at the three temperatures reflect that the inactivation rate constant increases with increasing temperature, and the higher the temperature, the greater the damage to the thalli. In order to achieve a better preservation effect, the freeze-dried powder of the Bayladdi yeast L2 can be preserved in a low-temperature environment with a low inactivation rate.
The logarithm (Lgk) of the inactivation rate constant of the lyophilized powder was plotted against the absolute temperature (1/T) according to equation 3, and the results are shown in FIG. 17 (b). From the trend line equation Lgk-5585.4/T +16.582, the inactivation rate constants of the lyophilized powder of Saccharomyces baylanicae L2 at 4 deg.C (refrigeration temperature) and-18 deg.C (refrigeration temperature) are assumed to be k 4 ＝2.62×10 -5 ，k -18 ＝4.77×10 -6 . The formula 3 can reversely deduce that the viable count of the freeze-dried powder of the saccharomyces bayanensis is 2.69 multiplied by 10 after being preserved for 1 year at 4 ℃ (refrigeration temperature) 9 CFU/g, viable count of 1.74 × 10 in 1 year of preservation at-18 deg.C (freezing storage temperature) 9 CFU/g。
b. Selenium-zinc-chromium-rich Bailadi yeast goat milk powder stability investigation
Mixing Balady yeast lyophilized powder and goat milk powder at a certain proportion, and estimating the shelf life of Balady yeast goat milk powder at refrigeration temperature (4 ℃) and normal temperature (25 ℃) according to the test method. The results are shown in FIG. 18, in which (a) the number of viable bacteria at different temperatures was varied with time; (b) arrhenius diagram.
As can be seen from fig. 18(a), after the freeze-dried powder and the goat milk powder are mixed uniformly according to a certain proportion, the inactivation rate constants of the freeze-dried powder and the goat milk powder at three test temperatures are respectively: k is a radical of 45 ＝0.0969(R 2 ＝0.9823)、k 50 ＝0.2052(R 2 ＝0.9806)、k 55 ＝0.3472(R 2 0.9904). According to the trend line equation determined in fig. 18 (b): Lgk-5785.9/T +17.196, the inactivation rate constants of Bayladida goat milk powder at 4 deg.C (refrigeration temperature) and 25 deg.C (room temperature) can be estimatedComprises the following steps: k is a radical of 4 ＝2.02×10 -4 、k 25 ＝4.03×10 -3 . Minimum viable count of 10 according to index requirements 6 CFU/g is the final value, the quality guarantee period of the Balady yeast goat milk powder is predicted through a first-order kinetic reaction equation (2), and the viable count of the Balady yeast goat milk powder can still reach 10 after the Balady yeast goat milk powder is stored for 473 days and 80 days at the temperature of 4 ℃ and 25 DEG C 6 CFU/g。
EXAMPLE 4 determination of tolerance of Ladida L2 in bacterial powder and goat milk powder to simulated gastrointestinal fluids
Completely dissolving 10g of goat milk powder in 90mL of sterile physiological saline, sucking 5mL of the goat milk powder by using a sterile syringe, adding the goat milk powder into 45mL of simulated gastric fluid (NaCl 9g/L, protease 3g/L and pH equal to 1.8), sampling once at intervals of 0.5h for viable count, and transferring 5mL to 45mL of simulated intestinal fluid (NaCl 9g/L, trypsin 20g/L, bile salt 3g/L and pH equal to 6.5) after reacting for 2h, and sampling once at intervals of 1h for viable count.
The viable count change of selenium-zinc-chromium Bailadi yeast L2 goat milk powder in simulated gastrointestinal fluid is shown in figure 19, wherein (a) is gastric fluid, and (b) is intestinal fluid.
As can be seen from figure 19, when the same addition amount of the bacteria powder is used as a reference and the added goat milk powder is used as a test group, the result shows that after the selenium-zinc-chromium-enriched Bailadding yeast L2 goat milk powder is treated in simulated gastric fluid for 2 hours, the number of the viable bacteria added to the goat milk powder is compared with that of a control group, which indicates that the probiotic bacteria powder has better survival ability in the simulated gastric fluid after being embedded in the goat milk powder. The viable count of the control group bacterial powder in the simulated gastric juice is obviously reduced, which shows that the bacterial powder without the protective agent is poor in resistance to the simulated gastric juice. The experimental group added with the goat milk powder in the bacterial powder has the advantage that the viable count of the goat milk powder is slowly reduced after the goat milk powder is treated in simulated gastric juice for 2 hours, which indicates that the goat milk powder has a protective effect on the goat milk powder in the simulated gastric juice.
And (3) transferring the sample treated in the gastric juice for 2 hours into simulated intestinal juice, and measuring the number of viable bacteria in the intestinal juice every 1 hour. The change trend of the viable count of the two samples is shown to increase and then decrease along with the increase of the digestion time, when the samples are digested in simulated intestinal fluid for 1 hour, the viable count of the two samples reaches the maximum, and the proliferation phenomenon probably occurs because some probiotics with the activity inhibited at low pH begin to growGradually recovering the activity. Jin et al report that free probiotic bacteria pass through gastric juice environment and then transfer to simulated intestinal fluid to proliferate, mainly when free probiotic bacteria are in strong acid environment, cell wall reinforcement of probiotic bacteria results in change of permeability of cell membrane, thereby changing H + Isolated outside the cell, thereby protecting the cell. The viable count of the Balady yeast L2 goat milk powder in simulated gastrointestinal fluid is obviously higher than that of the bacterial powder, which shows that the components of the goat milk powder such as protein, lactose, oligosaccharide and the like have good protection effect on thalli in the simulated gastrointestinal fluid, thereby showing good tolerance.
The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.
Claims (10)
1. A preparation method of nutrient-rich probiotic powder is characterized by comprising the following steps:
inoculating Balady yeast L2 into a culture medium, adding a selenium source, a zinc source and a chromium source, culturing, collecting bacterial sludge after culturing is finished, adding a freeze-drying protective agent into the bacterial sludge, and freeze-drying to obtain the nutrient-rich probiotic powder;
said Saccharomyces baylanicae L2 is deposited with the American type culture Collection under the accession number ATCC 9763.
2. The method of claim 1, wherein the selenium source, zinc source, and chromium source are sodium selenite, zinc sulfate, and chromium trichloride, respectively.
3. The preparation method of claim 1, wherein the concentration of the selenium source is 30 μ g/mL, the concentration of the zinc source is 300 μ g/mL, the concentration of the chromium source is 300 μ g/mL, the selenium content in the nutrient-enriched probiotic powder is 702.33-1400.91 μ g/g, the zinc content is 890.23-4193.18 μ g/g, the chromium content is 754.42-1700.78 μ g/g, and the amount of the bacteria is 10.75-28.20 g/L.
4. The method according to claim 1, wherein the culture conditions are: the culture time is 36-60h, the inoculation amount is 5-9%, and the pH is 5.0-6.0.
5. The preparation method of claim 1, wherein the lyoprotectant comprises the following components in percentage by mass: 4.5% of arginine, 7% of xylo-oligosaccharide, 24% of skim milk, 0.8% of sodium hydrogen phosphate and 5.5% of glycine.
6. The preparation method of claim 1, wherein the lyoprotectant is added in a mass ratio of 1:1 of the bacterial sludge to the lyoprotectant.
7. The nutrient-rich probiotic powder prepared by the preparation method according to any one of claims 1 to 6.
8. The use of the nutrient-enriched probiotic powder of claim 7 in goat milk powder.
9. A nutrient-rich probiotic goat milk powder, which contains the nutrient-rich probiotic powder of claim 8.
10. The nutrient-rich probiotic goat milk powder of claim 9, wherein the nutrient-rich probiotic goat milk powder is added in an amount of 0.05% -1%.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210834330.5A CN114958633B (en) | 2022-07-14 | 2022-07-14 | Preparation of nutrient-rich probiotic bacteria powder and application of nutrient-rich probiotic bacteria powder in goat milk powder |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210834330.5A CN114958633B (en) | 2022-07-14 | 2022-07-14 | Preparation of nutrient-rich probiotic bacteria powder and application of nutrient-rich probiotic bacteria powder in goat milk powder |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114958633A true CN114958633A (en) | 2022-08-30 |
| CN114958633B CN114958633B (en) | 2023-10-13 |
Family
ID=82968800
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210834330.5A Active CN114958633B (en) | 2022-07-14 | 2022-07-14 | Preparation of nutrient-rich probiotic bacteria powder and application of nutrient-rich probiotic bacteria powder in goat milk powder |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114958633B (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20050089530A1 (en) * | 2002-03-15 | 2005-04-28 | Pharma Nord Aps | Selenium yeast product, a method of preparing a selenium yeast product and the use of the product for preparing food, a dietary supplement or a drug |
| CN101045907A (en) * | 2006-03-30 | 2007-10-03 | 安琪酵母股份有限公司 | Selenium-rich and chromium-rich Saccharomyces cerevisiae, selenium-rich and chromium-rich yeast product and their production process |
| CN109527099A (en) * | 2018-12-28 | 2019-03-29 | 陕西科技大学 | A kind of fructus lycii compound probiotic goat milk piece and preparation method thereof |
| CN112195113A (en) * | 2020-10-12 | 2021-01-08 | 甘肃省科学院生物研究所 | Preparation method of saccharomycete enriched in iron, zinc and selenium |
| CN112457999A (en) * | 2020-11-10 | 2021-03-09 | 重庆蓝肽生物科技有限公司 | Selenium-rich saccharomyces cerevisiae strain and application thereof |
-
2022
- 2022-07-14 CN CN202210834330.5A patent/CN114958633B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20050089530A1 (en) * | 2002-03-15 | 2005-04-28 | Pharma Nord Aps | Selenium yeast product, a method of preparing a selenium yeast product and the use of the product for preparing food, a dietary supplement or a drug |
| CN101045907A (en) * | 2006-03-30 | 2007-10-03 | 安琪酵母股份有限公司 | Selenium-rich and chromium-rich Saccharomyces cerevisiae, selenium-rich and chromium-rich yeast product and their production process |
| CN109527099A (en) * | 2018-12-28 | 2019-03-29 | 陕西科技大学 | A kind of fructus lycii compound probiotic goat milk piece and preparation method thereof |
| CN112195113A (en) * | 2020-10-12 | 2021-01-08 | 甘肃省科学院生物研究所 | Preparation method of saccharomycete enriched in iron, zinc and selenium |
| CN112457999A (en) * | 2020-11-10 | 2021-03-09 | 重庆蓝肽生物科技有限公司 | Selenium-rich saccharomyces cerevisiae strain and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN114958633B (en) | 2023-10-13 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105018379B (en) | One plant of active lactobacillus plantarum of tool high anti-oxidation and its application | |
| CN106190893B (en) | The preparation method and application of one plant of lactobacillus fermenti for being suitable for vinegar brewing and its bacterium powder | |
| CN110106119B (en) | Lactobacillus rhamnosus M9 separated from breast milk and application thereof | |
| CN107988115B (en) | Lactobacillus plantarum and composite probiotic fermentation liquor and preparation method thereof | |
| CN101338283A (en) | Lactobacillus casei and applications thereof in solid-state fermentation | |
| CN113444664B (en) | Lactobacillus brevis for producing gamma-aminobutyric acid and application thereof | |
| CN114642686B (en) | Composite probiotics and its functions of delaying senility and resisting oxidation | |
| CN110577912A (en) | lactobacillus gasseri and application thereof in preparing fermented milk | |
| CN110257365A (en) | The preparation method and application of the immobilized microorganism microbial inoculum of kitchen garbage processing | |
| CN112126599B (en) | High-density culture method of lactobacillus helveticus, preparation of high-activity bacterium powder and application of high-density culture method | |
| CN110272846A (en) | Lactobacillus plantarum and its application with ABTS free radical and PTIO free radical scavenging activity | |
| CN106119152A (en) | The bacillus acidophilus of a kind of high-yield lactic acid rhzomorph and application thereof | |
| CN112574923A (en) | Streptococcus thermophilus capable of producing exopolysaccharides in high yield and application thereof | |
| CN115197870A (en) | Preparation method and application of lactobacillus with high antioxidant stress performance | |
| CN110257302B (en) | Screening method and application of lactobacillus strain with antioxidant capacity | |
| CN109810917B (en) | Lactobacillus salivarius and application thereof | |
| CN110607253B (en) | Streptococcus thermophilus and proliferation culture method and application thereof | |
| CN106119166B (en) | One plant of Switzerland lactic acid bacteria and its application | |
| CN114958633B (en) | Preparation of nutrient-rich probiotic bacteria powder and application of nutrient-rich probiotic bacteria powder in goat milk powder | |
| CN102220408B (en) | Lactic acid bacteria for producing bile salt hydrolase as well as screening method and application thereof | |
| CN116970512A (en) | Lactobacillus plantarum, and culture method and application thereof | |
| CN115029256B (en) | Kluyveromyces marxianus DPUL-F15 and application thereof | |
| CN111621461B (en) | Method for improving activity and tolerance of bacillus coagulans BC99 and application thereof | |
| CN114262680A (en) | Bacterial strain and application thereof | |
| CN105524864A (en) | Compound microbial inoculum product |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20230911 Address after: Room 1303, Building 3, Wanguanghui Business Center, Gongshu District, Hangzhou City, Zhejiang Province, 310000 Applicant after: Zhejiang Zhongmengchang Health Technology Co.,Ltd. Address before: 710021 Shaanxi city of Xi'an province Weiyang University Park Applicant before: SHAANXI University OF SCIENCE & TECHNOLOGY |
|
| TA01 | Transfer of patent application right | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CP03 | Change of name, title or address |
Address after: Room 1303, Building 3, Wanguanghui Business Center, Gongshu District, Hangzhou City, Zhejiang Province, 310000 Patentee after: Zhejiang Zhongmengchang Health Technology Co.,Ltd. Country or region after: China Address before: Room 1303, Building 3, Wanguanghui Business Center, Gongshu District, Hangzhou City, Zhejiang Province, 310000 Patentee before: Zhejiang Zhongmengchang Health Technology Co.,Ltd. Country or region before: China |