KR20050051432A - Feed additive for fish-breeding containing lactobacillus plantarum as effective ingradient - Google Patents

Abstract

The present invention relates to a feed for aquaculture feed fish comprising Lactobacillus plantarum ( Lactobacillus plantarum ) as an active ingredient, more specifically, acid resistance at pH 2.5, isolated from kimchi and resistant to bile in 400 ㎍ / ㎖ bile acid and A fish farming feed additive containing Lactobacillus plantarum, which is resistant to antibiotics against enolfloxacin, tyrosine and erythromycin and has fish growth promoting activity as an active ingredient. Feed additives containing Lactobacillus plantarum as an active ingredient, which are excellent in acid resistance, bile resistance and antibiotic resistance, and have an excellent effect on fish growth, can be usefully used for fish farming.

Description

Feed additive for fish-breeding containing Lactobacillus plantarum as effective ingradient}

The present invention relates to a feed additive for promoting animal growth using lactic acid bacteria as an active ingredient, and more particularly, to a feed for fish farming using an Lactobacillus strain having an acid resistance, bile resistance and antibiotic resistance as an active ingredient. will be.

Lactobacillus is a final metabolite, commonly referred to as lactic acid bacteria, which refers to bacteria that produce significant amounts of lactic acid. Lactobacillus is a kind of microorganisms that humans have been using for a long time, and is widely used in kimchi in Korea and dairy fermentation in the West.

In recent years, research on lactic acid bacteria has been actively conducted, and it is a very important industrial strain widely used in fermented milk production, health and functional foods, formal preparations, medicines, and probiotic products. Among the bacteria, Streptococcus sp. Caucus in (Pediococcus sp.), and the like Lou Kono in Stock (Leuconostoc sp.), genus Lactobacillus (Lactobacillus sp.), a genus Lactobacillus Spokane (Sporolactobacillus sp.) and Bifidobacterium (Bifidobacterium). In addition, lactobacillus, such as Lactobacillus, Pediococcus, Luconokstock, and Bisella genus, is the predominant species in Kimchi, a Korean fermented food product of Korea.

The lactic acid bacteria are gram-positive bacteria and have anaerobic or partial anaerobic properties. They are concentrated and settled in the gut gut wall of animals to decompose nutrients and fiber ingested by animals and use them as energy sources, and produce lactic acid and antibiotics. It is known to play an important role in maintaining intestinal health by inhibiting the growth of intestinal harmful bacteria.

In the case of larvae, the intestinal flora is not completely formed, so the growth of the larvae requires lactic acid bacteria, a strain that is beneficial for normal intestinal flora formation. In addition, lactic acid bacteria are also required to suppress the occurrence of diseases such as diarrhea caused by harmful strains such as Escherichia coli as the intestinal flora is destroyed by stress or absorption of harmful substances, travel and drinking. Lactobacillus is one of the predominant bacterial flora of the human intestinal microflora, and it has been reported to work beneficially, such as resistance to infection, anticancer effect, lowering cholesterol content in the blood and intestinal effect (Hentges, D., Hosono, A. , et al ., Agr. Biol. Chem., 1990, 54 (7), 1639-1643).

In addition, lactic acid bacteria have been used to promote animal growth and feed utilization, increase resistance to disease, inhibit the growth of harmful bacteria, reduce mortality, inhibit the production of toxic toxins, and produce various vitamins.

Because lactic acid bacteria use live bacteria themselves, their growth is inhibited by harsh environment in the human body when they are taken orally, i.e., an acidic environment where pH falls below 3, and digestive enzymes and bile acids secreted by the stomach and small intestine, thereby reducing the number of viable bacteria reaching the large intestine. In addition, there is a problem that the intestinal fixation rate is lowered. In addition, while reporting the efficacy according to the number of live bacteria of lactic acid bacteria, when the intake of commercially available lactobacillus fermented milk, the number of live bacteria of lactic acid bacteria that reach and colonize the colon is not enough to have a beneficial effect on the host (Roy , et al ., J. Dairy Sci. , 1990, 73, 1478-1484). In addition, it is urgent to select strains having excellent acid resistance in the preparation of bifidus fermented beverages (Berrada, et al ., J. Dairy Sci ., 1991, 14, 409-413).

As shown above, in order for the lactic acid bacteria to have a useful effect in humans or animals, the lactic acid bacteria introduced from the outside must reach the intestine without any disorder and adhere to the mucous membrane of the intestine, and then exhibit its function. To this end, it should be a strain that can be directly attached to the intestine, less destruction by gastric acid during oral administration, strong resistance to bile acids, and strong sterilizing ability of pathogens.

In Korean Patent Registration No. 142615, a new lactic acid bacterium having excellent acid resistance and cholesterol degradability was isolated from feces of infants in Korea, but the efficacy of lactic acid bacteria in livestock or human body was not reported.

In order to solve the above problems, the present inventors have isolated and identified Lactobacillus plantarum having excellent acid resistance, bile resistance and antibiotic resistance from kimchi, a Korean native vegetable fermented food, and the isolated lactic acid bacteria inhibited enteric harmful bacteria. The present invention has been completed by confirming that it promotes the growth of fish.

Accordingly, it is an object of the present invention to provide a fish farming feed additive containing lactic acid bacteria having excellent acid resistance, bile resistance and antibiotic resistance as an active ingredient.

In order to achieve the above object, the present invention provides a fish farming feed additive containing Lactobacillus Plantarum ( Lactobacillus Plantarum ) as an active ingredient excellent in acid resistance, bile resistance and antibiotic resistance.

Hereinafter, the present invention will be described in detail.

The present invention provides a feed additive for fish farming containing Lactobacillus Plantarum as an active ingredient excellent in acid resistance, bile resistance and antibiotic resistance.

The Lactobacillus plantarum, which is included in the fish food feed additive of the present invention, is acid resistant at pH 2.5, bile acid resistant at 400 μg / ml bile acid, and lactobacillus resistant to antibiotics against enolfloxacin, tyrosine, and erythromycin. It is preferable that it is plantarum, and it is more preferable that it is Lactobacillus plantarum J-124.

In the present invention, lactic acid bacteria having excellent acid resistance and bile resistance were isolated and selected using kimchi products sold in Korea and various kinds of kimchi produced at home. Selected lactic acid bacteria were classified on the basis of morphological, cultural and physiological characteristics and finally identified by sequencing of 16S rRNA.

The morphological and physiological characteristics of the isolated and screened lactic acid bacteria in the present invention are oval-shaped, Gram-positive, do not form spores, are not motile, and grow in both aerobic or anaerobic conditions. In addition, it oxidizes and fermentates, has no catalase or oxidase activity, grows in glucose medium, grows at 15 ° C. but does not grow at 45 ° C., but does not degrade esculine, but does not degrade arginine or gelatin, Do not reduce. It does not form indole or dextran, grows in 10% alcohol, produces DL-lactic acid, and occupies 43% of the G and C molecules in the sequence (see Table 2 ). In addition, the sugar availability of the lactic acid bacteria of the present invention, arabinos, cellobiose, esculin, fructose, glucose, galactose, lactose, maltose, mannitol, mannose, melezitose, raffinose, ribose, rhamnose, salinine, sorbitol, Sucrose, trehalose and xylose are used as sugars and cellulose and gluconate are not used as sugars (see Table 3 ).

As a result of 16S rDNA sequencing of the strain, the strain was identified as "Lactobacillus plantarum" having the nucleotide sequence of SEQ ID NO: 1 . In the present invention, the strain was named " Lactobacillus Plantarum J-124", and deposited on April 15, 2002 to KCTC (KCTC) in Korea Research Institute of Bioscience and Biotechnology (KRIBB). Number: KCTC 10224BP).

The Lactobacillus plantarum of the present invention did not survive at pH 1.5, but at pH 2.5, the viability was maintained at 90% or more until 60 minutes after treatment, and showed 50% or more at 90 minutes. In addition, at pH 3.5, the viability was maintained at 95% or more until 90 minutes after treatment, and 70% or more at 120 minutes (see Table 6 ). Therefore, the Lactobacillus plantarum of the present invention is excellent in viability even in a strong acid of pH 2.5, can survive in a harsh environment, that is, a strong acid environment in the human body after oral ingestion, it can have a beneficial function in vivo.

In addition, the Lactobacillus plantarum of the present invention can survive up to 10 ^5- fold dilution at a bile acid concentration of 100 μg / ml, and can survive up to 10 ^4- fold dilution at a bile acid concentration of 400 μg / ml, and have a bile acid concentration of 500 μg / ml. Survival up to 10 ^3- fold dilution in ml (see Table 7 ). Therefore, the Lactobacillus plantarum of the present invention is excellent in resistance to bile acids up to 500 ㎍ / ㎖ not be destroyed by bile acids secreted in the digestive tract after oral intake can have a beneficial function in vivo.

In addition, the Lactobacillus plantarum of the present invention has resistance to antibiotics such as enolfloxacin, tyrosine and erythromycin, and thus, such as enolfloxacin, tylosin or erythromycin. Grows in medium with antibiotics (see Table 8 ). The Lactobacillus plantarum of the present invention has resistance to antibiotics such as enolfloxacin, tyrosine and erythromycin, and thus, when the Lactobacillus plantarum of the present invention is mixed with an antibiotic, resistance to the antibiotic is given. Since the strain can survive without dying, the Lactobacillus plantarum of the present invention can be usefully used as a feed additive.

The Lactobacillus plantarum of the present invention is excellent in antibiotic resistance and can survive even with the addition of antibiotics, and in vivo, it is excellent in acid resistance and bile resistance, and thus the survival is maintained during reaching the intestine. Can function.

In addition, the Lactobacillus plantarum of the present invention can promote the growth of animals, in particular, the growth of fish, in a preferred embodiment of the present invention to prepare a feed additive containing the Lactobacillus plantarum J-124 strain as an active ingredient The weight of the flounder cultured using the feed prepared by adding the Lactobacillus plantarum strain of the present invention as compared to the flounder cultured with the fish for the flounder after adding it to the feed of the fish and the flounder cultured for general fish farming The growth rate was much better (see Table 5 ). Therefore, it can be seen that the Lactobacillus plantarum strain of the present invention can be usefully used for feed additives for fish farming.

In the aquaculture feed additive containing the Lactobacillus plantarum of the present invention, the fish is a group consisting of marine fish such as flounder, rockfish, red sea bream, sea bass, mullet, trout, and land fish such as eel, trout and mandarin fish. It is preferably selected from, and more preferably a flounder.

Fish feed additive for aquaculture containing Lactobacillus plantarum of the present invention preferably adds 10 ⁵ to 10 ¹⁰ or more, and 10 ⁶ to 10 ⁹ Lactobacillus plantarum per 1 g of feed More preferably.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Example 1 Lactobacillus Isolation and Identification

The present inventors separated the lactic acid bacteria using kimchi products that are commercially available in Korea and various kinds of kimchi produced at home.

After separating only the kimchi fermentation broth from Kimchi sample kimchi liquid ¹ ㎖ 10 1 to 10 ⁷ times stepwise diluted with a sterile physiological saline solution to, for each dilution 0.1 ㎖ emal S. (see Table 1 MRS, medium composition) in an agar medium The plate was incubated at 30 ° C. for 24 hours to isolate and select only lactic acid bacteria.

MS badge composition ingredient Concentration (g / ℓ) Proteos peptone 10 Beef Extract 10 Yeast extract 5 glucose 20 Twin 80 One Citric acid ammonium 2 Sodium acetate 5 Magnesium sulfate 0.1 Manganese sulfate 0.05 Sodium phosphate 2 Distilled water 1 ℓ

In order to identify the lactic acid bacteria selected above, the inventors investigated the biochemical properties of the lactic acid bacteria using a commercially available API kit (Bio-Reiux Inc., France).

As a result, the morphological and physiological characteristics of the lactic acid bacteria selected above are Gram-positive, do not form spores, do not move, grow in both aerobic or anaerobic conditions, catalase or oxidase as shown in Table 2 Does not produce, grows in glucose medium, grows at 15 ° C. but does not grow at 45 ° C., breaks down esculin, does not break down arginine or gelatin, does not reduce nitric acid, indole, VP or dextran Without forming it, grew in 10% alcohol, produced DL-lactic acid, and G and C molecules accounted for 43% in sequencing.

Morphological and Physiological Characteristics of Lactobacillus Plantarum Characteristics result Characteristics result Shape / Gram Oval. R / +. Oxidation / Fermentation Oxidation / Fermentation Spores - Esculin Decomposition + motility - Nitrate reduction - Growth in air + Arginine Degradation - Anaerobic + Gelatin breakdown - Catalase － Indole - Oxidase － Dextran formation - Glucose (acid) + Mol% G + C 43 Grow at 15 ℃ W Grown from 10% alcohol + Grow at 45 ℃ － DL-Lactic Acid +

In addition, the sugar availability of the lactic acid bacteria of the present invention, as shown in Table 3 , arabinose, cellobiose, esculin, fructose, glucose, galactose, lactose, maltose, mannitol, mannose, melezitose, raffinose, ribose, ram Noth, salicylic acid, sorbitol, sucrose, trehalose and xylose were used as sugars, and cellulose and gluconate were not used as sugars.

Sugar availability in Lactobacillus plantarum Party result Party result  Arabinose + Mannose + Cellulose - Melezitose + Cellobiose + Raffinose + Esculin + Ribose + Fructose + Rhamnose + Gluconate - Salincin + Glucose + Sorbitol + Galactose + Sucrose + Lactose + Trehalose + Maltose + Xylose + Mannitol +

Further, in order to identify the strains of the present inventors, 16S rDNA sequence was analyzed by an automatic sequence analyzer, and the strain was identified as Lactobacillus plantarum having the nucleotide sequence set forth in SEQ ID NO: 1 . The present inventors named the strain 'Lactobacillus plantarum J-124', and deposited it on the gene bank (KCTC) in Korea Research Institute of Bioscience and Biotechnology (KRIBB) as of April 15, 2002 (Accession No .: KCTC 10224). BP).

<Example 2> Change in body weight and body weight when Lactobacillus plantarum J-124 was administered to the flounder culture

The present inventors investigated the effect of Lactobacillus plantarum J-124 on the body size of the flounder when added to the feed used in the flounder culture.

Lactobacillus plantarum J-124 incubated for 18 hours at 30 ℃ in MS liquid medium and centrifuged to recover the microorganisms, suspended in 10% skim milk powder stabilizer so that the cell number is 10 ⁹ CFU / ㎖ and freeze To dry lactic acid bacteria powder was prepared. When the lactic acid bacteria powder was mixed with the powder and dispersed and administered to the flounder, it was mixed with the feed and used to make the final microbial count per 10 g of the feed to 10 ⁵ CFU / mL. In order to investigate the growth promoting effect in the flounder culture, the lactic acid bacteria of the present invention was administered for 90 days by repeating 300 fish of each group. At this time, the control group was administered to the fish feed group, and the experimental group was divided into the group administered with a mixture of Lactobacillus plantarum J-124 lactic acid powder to the fish feed, and then measured the body length and weight of the flounder.

As a result, when the Lactobacillus plantarum J-124 was administered to the flounder compared to the control group, the change in body length did not differ ( Table 4 ). However, as the body weight of Lactobacillus plantarum J-124 was increased, body weight gain increased significantly compared to the control group. As a result, when the last three months were administered, the growth rate of the experimental group was 33% higher than that of the control group ( Table 5 ). Therefore, it was confirmed that Lactobacillus plantarum J-124 of the present invention can effectively propagate the growth of fish, in particular halibut, can be useful as a feed additive for fish farming.

Measurement of Changes in the Length of Olive Flounder on Lactobacillus Planta Room J-124 Dosing period (month) Control Lactobacillus J-124 administration group Length (cm) Rate of increase ^* (%) Length (cm) % Increase 0 6.63 100 6.65 100 One 8.25 124 8.24 124 2 11.23 169 11.29 170 3 14.61 220 14.79 222

^* Growth rate = (length after initial administration / initial length) X 100

Measurement of Changes in Weight of Olive Flounder on Lactobacillus Planta Room J-124 Dosing period (month) Control Lactobacillus J-124 administration group Weight (g) Rate of increase ^* (%) Weight (g) % Increase 0 4.15 100 4.13 100 One 7.47 180 7.53 182 2 15.04 362 15.25 369 3 33.60 810 34.82 843

^* Increase = (weight after initial period / initial weight) x 100

Example 3 of Lactobacillus Planta Room J-124 Acid resistance measurement

We have found the ability of Lactobacillus plantarum J-124 to survive digestive enzymes such as gastric acid.

Specifically, Lactobacillus plantarum J-124 was inoculated in MS liquid medium and allowed to stand incubated at 30 ° C. for 12 hours so that the concentration of lactic acid bacteria was 10 ⁹ to 10 ¹⁰ CFU / mL. The cultured lactic acid bacteria were centrifuged at 6,000 rpm for 10 minutes to collect lactic acid bacteria, which were washed twice with sterile water, concentrated to 1/10 and used to have a cell number of about 10 ¹⁰ CFU / mL. Concentrated Lactobacillus plantarum J-124 was added to a sterile pH 2 buffer solution and placed in a 30 ° C. constant temperature water bath for 2 hours, then diluted in sterile water at a rate and smeared onto a plate of MS plate. The colony forming unit (CFU) was compared to indicate the viability of lactic acid bacteria, and the viability of lactic acid bacteria was calculated using Equation 1 below.

Viability (%) = A / B × 100

A: viable count after time elapsed, B: viable count at the beginning of the reaction

As a result, Lactobacillus plantarum J-124 did not survive at pH 1.5, and at pH 2.5 the viability was 92.98%, 92.98%, 56.14% and 31.58 over 30, 60, 90 and 120 minutes, respectively. Survival of% was shown, with survivability of at least 90% up to 60 minutes after treatment, and at least 50% viability at 90 minutes. In addition, at pH 3.5, the viability was 101.03%, 96.49%, 96.49%, and 71.93% over 30, 60, 90, and 120 minutes, respectively. It showed over 70% viability even in minutes ( Table 6 ).

Therefore, it can be seen that Lactobacillus plantarum J-124 of the present invention is a strain having excellent viability and excellent acid resistance even in a strong acid of pH 2.5 to pH 3.5.

Acid resistance measurement of Lactobacillus plantarum J-124 pH Viability according to processing time (minutes) 0 30 60 90 120 1.5 100 ^* 0 0 0 0 2.5 100 92.98 92.98 56.14 31.58 3.5 100 101.03 96.49 96.49 71.93

* Viability was expressed based on Equation (1).

Example 4 Measurement of Bile Resistance of Lactobacillus Planta Room J-124

The present inventors measured the viability of Lactobacillus plantarum J-124 against bile acids secreted in vivo.

Add the Lactobacillus plantarum J-124 to a solution containing 300 ㎍ / ㎖ of sterilized bile acid, put it in a 30 ° C constant temperature water bath, leave for 2 hours, dilute it in sterile water at a rate and smear it on MS flat plate By comparing the number of colony forming unit (colony forming unit, CFU) was shown the viability of the lactic acid bacteria.

As a result, as the 10-fold diluted in the culture medium are the bile acid is not added as controls, Lactobacillus Planta room J-124 number of colonies by the reduction of 10 if ^7-fold dilution showing a mean 3.5 number of colonies, 100 ㎍ / ㎖ the bile acid concentration reduction in the number of colonies as the 10-fold dilution when diluted 10 ⁵ times showed the average number of colonies. At 200 µg / ml, 300 µg / ml, and 400 µg / ml bile acid concentrations, colony counts decreased with 10-fold dilution, resulting in an average of 1.5, 1, and 0.5 colonies, respectively, at 10- and ^4- fold dilutions. At the bile acid concentration, colony count decreased with 10-fold dilution, and averaged 2.5 colony counts after diluting 10 ^3- fold ( Table 7 ).

Through the above results, Lactobacillus plantarum J-124 can survive up to 10 ⁵ fold dilution at a bile acid concentration of 100 μg / ml, and can survive up to 10 ⁴ fold dilution at a bile acid concentration of 200 to 400 μg / ml, It can be seen that it can survive up to 10 ^3- fold dilution at a bile acid concentration of 500 μg / ml.

Thus, Lactobacillus plantarum J-124 can survive at a bile acid concentration of 100 to 400 μg / ml, at a higher concentration of 200 to 400 μg / ml and at the highest 500 μg / ml It can be seen that the biliary resistance is excellent.

Bile Resistance Measurements of Lactobacillus Planta Room J-124 Bile Acid Concentration (µg / mL) Dilution factor 10 ^-1 10 ^-2 10 ^-3 10 ^-4 10 ^-5 10 ^-6 10 ^-7 0 > > > > >   74 81    4 3 100 > >   60 61   10 6    2 0 0 - 200 >   38 44    7 1    1 2 - - - 300 >   12 16    3 5    2 0 0 - - 400 >   11 19    2 in 1    1 0 0 - - 500 > >    4 1    0 0 0 -

>: Indicates that there are many colonies and cannot count

-: It shows no colony number.

Example 5 Antibiotic Resistance Measurement of Lactobacillus Planta Room J-124

We measured whether Lactobacillus plantarum J-124 is resistant to antibiotics. Antibiotic resistance experiments were carried out by treating each antibiotic solution (30 μg) sterilized with a microfilter (0.2 μm) on a sheet of paper and drying, followed by 10 ml of 0.7% soft agar containing 1% test bacteria. The plate was placed on an S aga plate and incubated at 30 ° C. for 48 hours to determine the size of the transparent ring around the plate.

As a result, an 11 mm clear ring was formed around enolfloxacin, a 12 mm clear ring around tyrosine, and a 13 mm clear ring around erythromycin ( Table 8 ).

Thus, it can be seen that the Lactobacillus plantarum J-124 of the present invention is resistant to the antibiotics enolfloxacin, tyrosine and erythromycin.

Antibiotic Resistance of Lactobacillus Planta Room J-124 Antibiotic (30 µg / plate) Size of transparent ring (mm) Zinc bacitracin - Oxytetracycline - Colistin - Corotetracyclin - Enolfloxacin 11 Tylosin 12 Gentamicin - Erytromycin 13 Oxytetracycline - Separexin (Cephalexin) -

-: Means that no transparent ring is formed.

As described above, the Lactobacillus plantarum of the present invention is excellent in acid resistance, bile resistance, and antibiotic resistance, exhibits an excellent effect on the intestinal pathogenic bacterium inhibiting ability and the beneficial flora increase in the intestine, and an excellent effect on the flounder growth. It can be usefully used as a feed additive for fish farming.

<110> Korea Research Institute of Bioscience and Biotechnology <120> Feed additive for fish-breeding containing Lactobacillus plantarum as effective ingradient <130> 3p-11-31 <160> 1 <170> KopatentIn 1.71 <210> 1 <211> 705 <212> DNA <213> Lactobacillus Plantarum J-124 <400> 1 cgaacgctgc cggcgtgcct aatacatgca agtcgaacga actctggtat tgattggtgc 60 ttgcatcatg atttacattt gagtgagtgg cgaactggtg agtaacacgt gggaaacctg 120 cccagaagcg ggggataaca cctggaaaca gatgctaata ccgcataaca acttggaccg 180 catggtccga gcttgaaaga tggcttcggc tatcactttt ggatggtccc gcggcgtatt 240 agctagatgg tggggtaacg gctcaccatg gcaatgatac gtagccgacc tgagagggta 300 atcggccaca ttgggactga gacacggccc aaactcctac gggaggcagc agtagggaat 360 cttccacaat ggacgaaagt ctgatggagc aacgccgcgt gagtgaagaa gggtttcggc 420 tcgtaaaact ctgttgttaa agaagaacat atctgagagt aactgttcag gtattgacgg 480 tatttaacca gaaagccacg gctaactacg tgccagcagc cgcggtaata cgtaggtggc 540 aagcgttgtc cggatttatt gggcgtaaag cgagcgcagg cggtttttta agtctgatgt 600 gaaagccttc ggctcaaccg aagaagtgca tcggaaactg ggaaacttga gtgcagaaga 660 ggacagtgga actccatgtg tagcggtgaa atgcgtagat atatg 705

Claims (5)

A fish farming feed additive containing Lactobacillus Plantarum as an active ingredient having acid resistance, bile resistance and antibiotic resistance. The fish farming feed additive according to claim 1, wherein the Lactobacillus plantarum is Lactobacillus plantarum J-124 (Accession Number: KCTC 10224BP). The fish feed additive according to claim 1, wherein the fish is selected from the group consisting of marine fish such as halibut, rockfish, red snapper, freshfish, mullet, and trout, and land fish such as eel, trout and mandarin. 4. The fish farming feed additive according to claim 3, wherein the fish is a flounder. The method of claim 1, wherein the feed additive Lactobacillus Planta room feed additive for fish farming, characterized in that the feed 1 10 ⁵ to 10 ¹⁰ per g contained in.