Microbial fermentation of botanicals

Abstract

A method for microbial fermentation of botanicals includes steps of: fermenting with wall-breaking fungi, and then fermenting with probiotics. The wall-breaking fungi are wood-grown fungi and/or Cordyceps. The method can effectively destroy the cell wall of the botanical, promote release of effective ingredients of the botanical and improve utilization rate of the botanical. In addition, due to fermenting with the wall-breaking fungi, the botanical medicine also contains the active ingredients of the wall-breaking fungi. Thereby the efficacy of the botanical is increased.

Claims

1. A microbial fermentation method of botanicals, comprising steps of: weighing 10 Kg of ginseng, pulverizing to 30 meshes, pouring into a fermenting tank, adding 80 Kg of culture solution which, by mass ratio, comprises: glucose 2.4%, yeast extract 0.2%, peptone 0.4%, potassium dihydrogen phosphate 0.1%, magnesium sulfate 0.1%, and distilled water 96.8%; thoroughly mixing, adjusting pH to 6.8 with sodium hydroxide solution, sterilizing with steam for 30 min; cooling to room temperature, and inoculating Ganoderma lucidum, keeping a stirring speed in the fermentation tank at 120 rpm, keeping a temperature at 20° C., and fermenting for 15 days; then adding 1‰ total raw material mass fraction of yeast and 1‰ total raw material mass fraction of Lactobacillus, and statically fermenting at 28° C. for 7 days; then adding 1‰ total raw material mass fraction of Acetobacterium, statically fermenting at 30° C. for 7 days; filtering and collecting liquid; wherein the yeast is Pichia ohmeri, CGMCC No. 2.1803; the Lactobacillus is Lactobacillus plantarum, CGMCC No. 1.6971, with millions of viable bacteria; and the Acetobacterium is Acetobacter pasteurianus CGMCC No. 1.41, with millions of viable bacteria.

2. A microbial fermentation method of botanicals, comprising steps of: weighing 10 Kg of ginseng, pulverizing to 30 meshes, pouring into a fermenting tank, adding 80 Kg of culture solution which, by mass ratio, comprises: glucose 2.4%, yeast extract 0.2%, peptone 0.4%, potassium dihydrogen phosphate 0.1%, magnesium sulfate 0.1%, and distilled water 96.8%; thoroughly mixing, adjusting pH to 6.8 with sodium hydroxide solution, sterilizing with steam for 30 min; cooling to room temperature, and inoculating Ganoderma, keeping a stirring speed in the fermentation tank at 100 rpm, keeping a temperature at 23° C., and fermenting for 15 days; then adding 1‰ total raw material mass fraction of yeast and 1‰ total raw material mass fraction of Lactobacillus, and statically fermenting at 28° C. for 7 days; then adding 1‰ total raw material mass fraction of Acetobacterium, statically fermenting at 30° C. for 7 days; filtering and collecting liquid; wherein the Ganoderma is Ganoderma tsugae, CGMCC No. 5.772; the yeast is Saccharomyces cerevisiae, CGMCC No. 2.3888; the Lactobacillus is Lactobacillus panis, CGMCC No. 1.3925, with millions of viable bacteria; and the Acetobacterium is Acetobacter pasteurianus CGMCC No. 1.41, with millions of viable bacteria.

3. A microbial fermentation method of botanicals, comprising steps of: weighing 10 Kg of pueraria, pulverizing to 20 meshes, pouring into a fermenting tank, adding 100 Kg of culture solution which, by mass ratio, comprises: glucose 2.4%, yeast extract 0.2%, peptone 0.4%, potassium dihydrogen phosphate 0.1%, magnesium sulfate 0.1%, and distilled water 96.8%; thoroughly mixing, adjusting pH to 6.5 with sodium hydroxide solution, sterilizing at 121° C. for 30 min; cooling to room temperature, and inoculating Cordyceps, keeping a stirring speed in the fermentation tank at 150 rpm, keeping a temperature at 25° C., and fermenting for 10 days; then adding 3% total raw material mass fraction of yeast and 1‰ total raw material mass fraction of Lactobacillus, and statically fermenting at 28° C. for 8 days; then adding 1‰ total raw material mass fraction of Acetobacterium, statically fermenting at 30° C. for 7 days; filtering and collecting liquid; wherein the yeast is Saccharomyces cerevisiae, CGMCC No. 2.3973; the Lactobacillus is Lactobacillus buchneri, CGMCC No. 1.3114, with millions of viable bacteria; and the Acetobacterium is Acetobacter pasteuranus SHBCC D24822, with millions of viable bacteria.

Description

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

(1) The following embodiments are intended to describe the present invention in detail, and are not intended to limit the scope of the present invention. Modifications and adjustments made by those skilled in the art based on the disclosure also belong to the scope of the present invention.

Embodiment 1

(2) Weighing 10 Kg of ginseng, pulverizing to 30 meshes, pouring into a fermenting tank, adding 80 Kg of culture solution (by mass ratio: glucose 2.4%, yeast extract 0.2%, peptone 0.4%, potassium dihydrogen phosphate 0.1%, magnesium sulfate 0.1%, and distilled water 96.8%), thoroughly mixing, adjusting pH to 6.8 with sodium hydroxide solution, sterilizing with steam for 30 min; cooling to room temperature, and inoculating Ganoderma lucidum (Xiangchizhi No. 1, Hunan Province non-major crop variety registration certificate number XPD010-2013, obtained from State Key Laboratory of Sub-health Intervention Technology), keeping a stirring speed in the fermentation tank at 120 rpm, keeping the temperature at 20° C., and fermenting for 15 days; then adding 1‰ (total raw material mass fraction) yeast (Pichia ohmeri, CGMCC No. 2.1803, millions of viable bacteria) and 1‰ (total raw material mass fraction) Lactobacillus (Lactobacillus plantarum, CGMCC No. 1.6971, millions of viable bacteria), and statically fermenting at 28° C. for 7 days; then adding 1‰ (total raw material mass fraction) Acetobacterium (Acetobacter pasteurianus CGMCC No. 1.41, purchased from Shanghai No. 1 Brewing Plant, millions of viable bacteria), statically fermenting at 30° C. for 7 days; filtering and collecting liquid.

(3) According to test, the ginseng liquid obtained by the present invention has a total ginsenoside content of 1.06 mg/mL (the determination method refers to that in the paper of “Content determination of total ginsenoside, Gao Liping et al., Journal of Zhejiang Institute of Engineering, 2012, 31(3): 382-388”), which is 7.01 times higher than the conventional decoction (a weight ratio of ginseng and water is 1:8). Furthermore, 0.75 mg/mL Ganoderma triterpenoid content is also detected in the liquid (the assay method refers to that in “Chinese Pharmacopoeia 2015 edition”). All data are average values of triplicate samples.

Embodiment 2

(4) Weighing 10 Kg of ginseng, pulverizing to 30 meshes, pouring into a fermenting tank, adding 80 Kg of the culture solution as mentioned in the embodiment 1, thoroughly mixing, adjusting pH to 6.8 with sodium hydroxide solution, sterilizing with steam for 30 min; cooling to room temperature, and inoculating Ganoderma (Ganoderma tsugae, CGMCC No. 5.772, belonging to Ganoderma sinense), keeping a stirring speed in the fermentation tank at 100 rpm, keeping temperature at 23° C., and fermenting for 15 days; then adding 1‰ (total raw material mass fraction) yeast (Saccharomyces cerevisiae, CGMCC No. 2.3888, millions of viable bacteria) and 1‰ (total raw material mass fraction) Lactobacillus (Lactobacillus panis, CGMCC No. 1.3925, millions of viable bacteria), and statically fermenting at 28° C. for 7 days; then adding 1‰ (total raw material mass fraction) Acetobacterium (Acetobacter pasteurianus CGMCC No. 1.41, millions of viable bacteria), statically fermenting at 30° C. for 7 days; filtering and collecting liquid.

(5) According to test, the ginseng liquid obtained by the present invention has a total ginsenoside content of 1.03 mg/mL (the determination method as mentioned in the embodiment 1), which is 6.81 times higher than the conventional decoction (a weight ratio of ginseng and water is 1:8). Furthermore, 0.64 mg/mL Ganoderma triterpenoid content is also detected in the liquid (the assay method as mentioned in the embodiment 1). All data are average values of triplicate samples.

Embodiment 3

(6) Weighing 10 Kg of pueraria, pulverizing to 20 meshes, pouring into a fermenting tank, adding 100 Kg of culture solution as mentioned in the embodiment 1, thoroughly mixing, adjusting pH to 6.5 with sodium hydroxide solution, sterilizing at 121° C. for 30 min; cooling to room temperature, and inoculating Cordyceps (Xiangbeichongcao No. 1, Hunan Province non-major crop variety registration certificate number XPD009-2013, obtained from State Key Laboratory of Sub-health Intervention Technology), keeping a stirring speed in the fermentation tank at 150 rpm, keeping temperature at 25° C., and fermenting for 10 days; then adding 3‰ (total raw material mass fraction) yeast (Saccharomyces cerevisiae, CGMCC No. 2.3973, millions of viable bacteria) and 1‰ (total raw material mass fraction) Lactobacillus (Lactobacillus buchneri, CGMCC No. 1.3114, millions of viable bacteria), and statically fermenting at 28° C. for 8 days; then adding 1‰ (total raw material mass fraction) Acetobacterium (Acetobacter pasteuranus SHBCC D24822, millions of viable bacteria), statically fermenting at 30° C. for 7 days; filtering and collecting liquid.

(7) According to test, the pueraria liquid obtained by the present invention has a puerarin content of 0.119 mg/mL (the determination method refers to that in “Health Foods of Chinese National Standard GB/T 22251-2008”), which is 1.25 times higher than the conventional decoction (a weight ratio of pueraria and water is 1:10). Furthermore, 11.3 μg/mL cordycepin content is also detected in the liquid (the assay method refers to that in “Screening of high yield strains of cordycepin and effects of different additives on the yield of cordycepin, Wang Lei et al., Journal of Fungal Materials, 2012, 31(3): 382-388”). All data are average values of triplicate samples.

Embodiment 4

(8) Weighing 10 Kg of pueraria, pulverizing to 20 meshes, pouring into a fermenting tank, adding 100 Kg of the culture solution as mentioned in the embodiment 1, thoroughly mixing, adjusting pH to 6.5 with sodium hydroxide solution, sterilizing at 121° C. for 30 min; cooling to room temperature, and inoculating Cordyceps (Cordyceps militaris, CGMCC No. 5.856), keeping a stirring speed in the fermentation tank at 130 rpm, keeping temperature at 28° C., and fermenting for 10 days; then adding 3‰ (total raw material mass fraction) yeast (Pichia membranaefaciens, CGMCC No. 2.661, millions of viable bacteria) and 1‰ (total raw material mass fraction) Lactobacillus (Lactobacillus panis, CGMCC No. 1.3925, millions of viable bacteria), and tatically fermenting at 28° C. for 8 days; then adding 1‰ (total raw material mass fraction) Acetobacterium (Acetobacterium Huniang 1.01, millions of viable bacteria), statically fermenting at 30° C. for 7 days; filtering and collecting liquid.

(9) According to test, the pueraria liquid obtained by the present invention has a puerarin content of 0.113 mg/mL (the determination method as mentioned in the embodiment 3), which is 1.19 times higher than the conventional decoction (a weight ratio of pueraria and water is 1:10). Furthermore, 13.7 μg/mL cordycepin content is also detected in the liquid (the assay method as mentioned in the embodiment 3). All data are average values of triplicate samples.

(10) The following biological materials are readily accessible to the public, and have been deposited under the terms of the Budapest Treaty with the China General Microbiological Culture Collection Center (CGMCC), Institute of Microbiology, Chinese Academy of Sciences, Haidian, Beijing 100080, China:

(11) (1) Name: Pichia ohmeri

(12) Accession Number: CGMCC No. 2.1803

(13) Date of Deposit: 1994 Oct. 1

(14) Classification: Ascomycota.genus, Pichia sp, P. ohmeri

(15) Origin: China

(16) (2) Name: Lactobacillus plantarum

(17) Accession Number: CGMCC No. 1.6971

(18) Date of Deposit: 2007 Jul. 1

(19) Classification: Bacteria, Firmicutes, Bacilli, Lactobacillales, Lactobacillaceae, Lactobacillus

(20) Origin: China

(21) (3) Name: Acetobacter pasteurianus

(22) Accession Number: CGMCC No. 1.41

(23) Date of Deposit: 1950 Dec. 1

(24) Classification: Proteobacteria, Acetobacter, Acetobacter pasteurianus

(25) Origin: China

(26) (4) Name: Ganoderma tsugae

(27) Accession Number: CGMCC No. 5772

(28) Date of Deposit: 1999 Jun. 22

(29) Classification: Basidiomyceta, Lamella, Pales, Poramellaceae, Ganoderma

(30) Origin: Holland

(31) (5) Name: Saccharomyces cerevisiae

(32) Accession Number: CGMCC No. 2.3888

(33) Date of Deposit: Publicly available

(34) Classification: Eukarya, Fungi, Ascomycota, Hemiascomycete, Saccharomycetales, Saccharomycetaceae, Saccharomyces, S. cerevisiae

(35) Origin: China

(36) (6) Name: Lactobacillus panis

(37) Accession Number: CGMCC No. 1.3925

(38) Date of Deposit: 2005 Apr. 25

(39) Classification: Bacterium, Firmicutes, Bacillus, Lactobacillus, Lactobacillaceae, Lactobacillus panis

(40) Origin: China

(41) (7) Name: Saccharomyces cerevisiae

(42) Accession Number: CGMCC No. 2.3973

(43) Date of Deposit: 2008, Sep. 16

(44) Classification: Eukarya, Fungi, Ascomycota, Hemiascomycete, Saccharomycetales, Saccharomycetaceae, Saccharomyces, S. cerevisiae

(45) Origin: China

(46) (8) Name: Lactobacillus buchneri

(47) Accession Number: CGMCC No. 1.3114

(48) Date of Deposit: 2002 Jul. 2

(49) Classification: Bacterium, Firmicutes, Bacillus, Lactobacillus, Lactobacillaceae, Lactobacillus buchneri

(50) Origin: China

(51) The following biological material is readily accessible to the public, and has been deposited with the Shanghai Bioresource Collection Center (SHBCC), Fengxian, Shanghai, 201414, China:

(52) (9) Name: Acetobacter pasteurianus

(53) Accession Number: SHBCC D24822

(54) Date of Deposit: 1971 Dec. 1

(55) Classification: Proteobacteria, Acetobacter, Acetobacter pasteurianus

(56) Origin: China