DEVRIES ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 88, NO. 1, 2005 5 DIETARY SUPPLEMENTS
Microbiological Assay-Trienzyme Procedure for Total Folates in Cereals and Cereal Foods: Collaborative Study JONATHAN W. DEVRIES Medallion Laboratories, General Mills Inc., 9000 Plymouth Ave North, Minneapolis, MN 55427 JEANNE I. RADER U.S. Food and Drug Administration, Center for Food Safety and Nutrition, 5100 Paint Branch Pkwy, College Park, MD 20740-3835 PAMELA M. KEAGY and CAROL A. HUDSON U.S. Department of Agriculture, Western Regional Research Center, 800 Buchanan St, Albany, CA 94710 Collaborators: G. Angyal; J. Arcot; M. Castelli; N. Doreanu; C. Hudson; P. Lawrence; J. Martin; R. Peace; L. Rosner; H.S. Strandler; J. Szpylka; H. van den Berg; C. Wo; C. Wurz
In 1996, U.S. Food and Drug Administration regulations mandated the fortification of enriched cereal-grain products with folic acid, thereby emphasizing the need for validated methods for total folates in foods, particularly cereal products. The AOAC Official Methods (944.12, 960.46) currently used for the analysis of folate in foods for compliance purposes are microbiological methods. When the fortification regulations were finalized, no Official AOAC or Approved AACC methods for folate in cereal-grain products were in place. The AOAC Official Method (992.05) for folic acid in infant formula does not incorporate important improvements in the extraction procedure and was not considered suitable for the analysis of folates in foods in general. A microbiological assay protocol using a trienzyme extraction procedure was prepared and submitted for comments to 40 laboratories with recognized experience in folate analysis. On the basis of comments, the method was revised to have the conjugase (gamma-glutamyl-carboxy-peptidase) treatment follow a protease treatment, to include the use of cryoprotected inoculum, and to include the spectroscopic standardization of the standard and optional use of microtiter plates. Thirteen laboratories participated in a collaborative study of 10 required and 10 optional cereal-grain products, including flour, bread, cookies, baking mixes, and ready-to-eat breakfast cereals. The majority of the participating laboratories performed the assay by Submitted for publication August 2004. The recommendation was approved by the Methods Committee on Commodity Foods and Commodity Products as First Action. See “Official Methods Program Actions,” (2004) Inside Laboratory Management, July/August issue. Corresponding author’s e-mail:
[email protected].
the standard test tube method; others used the microtiter plate modification for endpoint quantitation with equal success. For the required products, the relative standard deviation between laboratories (RSDR) ranged from 7.4 to 21.6% for 8 fortified (or enriched) products compared with expected (Horwitz equation-based) values of 11–20%. RSDR values were higher (22.7–52.9%) for 2 unfortified cereal-grain products. For the optional products, the RSDR ranged from 1.8 to 11.2% for 8 fortified products. RSDR values were higher (27.9–28.7%) for 2 unfortified cereal-grain products. Based on the results of the collaborative study, the microbiological assay with trienzyme extraction is recommended for adoption as Official First Action.
F
olate and its polyglutamyl homologs (Figure 1) are essential vitamins for humans. Naturally occurring folates are reduced derivatives; fully oxidized folic acid is only found in the diet when foodstuffs are fortified or enriched (i.e., supplemented with folic acid) or when naturally occurring folates are oxidized. Deficiency of folate leads to megaloblastic anemia and a general impairment of cell division that is more apparent in tissues that turn over rapidly (e.g., the hematopoietic system and cells lining the digestive tract). Weakness, tiredness, diarrhea, and anorexia are associated with folate deficiency. Recent studies have provided evidence that folate supplementation reduces the incidence of neural tube defects in certain high-risk populations (1–4). Studies are currently in progress to determine whether folate deficiency per se may contribute to the development of atherosclerosis (5, 6). In 1996, the U.S. Food and Drug Administration (FDA) finalized regulations mandating the fortification of enriched cereal-grain products with folic acid. Accurate measurement of folate content of foods is essential for purposes of food labeling and for the development of databases of folate content of foods.
6 DEVRIES ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 88, NO. 1, 2005
Figure 1. Folic acid structure (various forms available in the diet).
For food labeling and database purposes, folates have traditionally been analyzed with microbiological assays, such as AOAC Official Methods 944.12, 992.05, and 960.46. AOAC Official Method 992.05 uses a conjugase enzyme treatment during extraction for conversion of the polyglutamyl vitamers to folic acid. Advances in folate methodology were made by a number of workers (7–13). In 1990, DeSouza and Eitenmiller (14) showed that the use of protease and amylase in addition to the conjugase significantly increased the measured level of folate in foods. Also in 1990, Martin et al. (15) demonstrated the broad applicability of the trienzyme (conjugase, protease, and amylase) methodology to a wide variety of foods. Other researchers (16–19) further demonstrated the application of the trienzyme system. Finally, in 1998, Rader et al. (20) found that digesting the test portion with protease followed by a protease deactivation step before treatment with the other enzymes resulted in the highest measured folate level (Table 1). Laboratories performing the basic AOAC Official Methods were applying the trienzyme procedure in a variety of ways, including the use of microtiter plates instead of test tubes for the determinative Lactobacillus casei growth and turbidity measurement steps. Although the microbiological assay for folate was used for several decades, significant differences in scientific opinion existed regarding the optimum procedures. To ascertain that the optimum method would be collaboratively studied, a complete review of the literature was undertaken, a method based on the review was drafted in AOAC format, and input from analysts active in folate analysis was sought. The method was submitted to 40 laboratories active in folate analysis for comment and feedback. The written method was revised and the microtiter plate end point measurement option was included. Based on the comments of the laboratories, the final version of the method intended for collaborative study was prepared.
Collaborative Study For the collaborative study, cereal-based products with a range of protein, fat, and carbohydrate (fiber and starch) were selected (Table 2). Products were selected to cover a wide range of folate levels, including both fortified and unfortified products. Results from individual test samples were used to determine overall variability (SDR, R, and RSDR). Twenty different test samples were prepared by grinding, homogenizing, sealing in glass jars, and encoding. All food products for the collaborative study were purchased at local supermarkets in quantities adequate to ensure that homogeneous test samples would be supplied to all collaborators. After grinding (if necessary), test samples were mixed to ensure homogeneity, sealed in glass jars, encoded, and placed in the dark for storage until shipment. Collaborators were provided with instructions to analyze individual test samples and report the results. Collaborators were provided with 2 sets of test samples: one set of 10 was labeled “required” and were to be analyzed first. The second set was labeled “optional” to allow the laboratory to analyze them as time and
Table 1. Effect of sequence of enzyme pretreatments, mg/100 g test sample found Product
Conjugase
Trienzyme, conjugase first
Trienzyme, protease first
All-purpose flour
26.1 ± 1.25
33.1 ± 0.3
41.3 ± 1.4
Rotelle
31.4 ± 1.65
39.6 ± 1.3
49.6 ± 2.2
Long-grain and wild rice
50.7 ± 8.3
59.4 ± 1.3
71.1 ± 3.5
629.3 ± 48.05
677.8 ± 52.2
665.8 ± 29.6
Bran cereal
DEVRIES ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 88, NO. 1, 2005 7 Table 2. Characteristics of samples tested, percentage makeup Product
Solids
Protein
Fat
Carbohydrate
Fiber
Wheat flour, fortified white
88.1
10.3
1.0
76.3
2.7
Wheat flour, unfortified white
88.1
10.3
1.0
76.3
2.7
Wheat flour, whole wheat
89.7
13.7
1.9
72.6
12.6
White bread from wheat flour
63.3
8.2
3.6
49.5
2.3
Macaroni, dry
89.7
12.8
1.6
74.7
2.4
Cookies, low-fat, gingersnap
94.7
5.6
9.8
76.9
Cookies, fat-free oatmeal
87.5
5.9
1.5
78.6
7.3
Cookies, high-fat shortbread
96.3
6.1
24.4
64.5
1.6
Cookies, oatmeal, regular
94.3
6.2
18.1
68.7
3.1
a
RTE cereal, corn flakes
97.4
8.1
0.3
86.1
6.0
RTE cereal, extruded oat
95.0
15.1
6.4
69.1
12.0
RTE cereal, wheat flakes
95.4
9.5
1.7
9.5
10.0
RTE cereal, crispy rice
97.5
6.4
0.4
88.6
1.4
RTE cereal, extruded bran
97.0
14.3
1.8
74.4
29.9
Pancake mix, low-fat
90.9
10.0
1.7
73.6
2.7
Biscuit mix, high-fat
90.8
8.0
15.4
63.3
2.3
Tortillas
55.9
5.7
2.5
46.6
5.2
Corn meal
88.4
8.5
1.7
77.7
5.2
a
RTE = Ready-to-eat.
resources allowed. Laboratories were given the option of using the test tube method, the microtiter plate method, or both. Because laboratories conducting this study were generally familiar with microbiological assays, no practice samples were supplied. Laboratories were encouraged, but not required, to purchase and include NIST SRM 1846 as a reference material for the study. SRM 1846, available from the National Institutes of Standards and Technology (NIST), is a spray-dried, milk-based infant formula. The mass fraction value for folic acid in this material is 1.29 ± 0.28 mg/kg. SRM 1846 was also included as an unknown test sample among those sent as part of the required examination set. AOAC Official Method 2004.05 Total Folates in Cereals and Cereal Foods Microbiological Assay-Trienzyme Procedure First Action 2004
(Applicable to cereal grains and cereal grain foods containing added folate (folic acid) or natural occurring folates with levels from 7.6 mg/100 g to 100% folate.) See Table 2004.05A for the results of the interlaboratory study supporting acceptance of the method. Caution: See Appendix B for laboratory safety. (1) Ammonium hydroxide. (2) Hydrochloric acid. (3) Potassium or sodium hydroxide: Extremely caustic; can cause severe burns; protect skin and eyes. (4) Flammable liquids: Perform operations
behind safety barrier with hot water, steam, or electric mantle heating. Use effective fume removal device to remove flammable vapors as produced. Leave ample headroom in flask and add boiling chips before heating is begun. All controls, unless vapor sealed, should be located outside of vapor area. (5) Toxic liquids: Use effective fume removal device to remove vapors as produced. Avoid contact with skin. Method uses the organism Lactobacillus casei subspecies rhamnosus-ATCC 7469 to determine the amount of folate present in foods and vitamin concentrates. Method can also be semiautomated through the use of automated dilution and turbidity reading instruments or the 96 well microtiter plate and reader system. Note: Folates are light and oxygen sensitive. Use of yellow lighting and low actinic glassware is recommended. Preparation and storage of samples under subdued lighting is essential. A. Principle Foods, with water added, are autoclaved to break up particles, gelatinize starch, destroy extraneous microorganisms, and denature proteins to enhance enzymatic attack and make folate more extractable. Folate (pteroylglutamic acid in various forms) occurs naturally in foods bound to glutamic acid residues of varying chain lengths. Most naturally occurring folates cannot be used by the assay organism. Folic acid
8 DEVRIES ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 88, NO. 1, 2005 Table 2004.05A. Interlaboratory study results of trienzyme method for determination of folate in cereal grains and grain products No. of labsa
Mean, mg/100 g
Flour, fortified
13(1)
134.1
15.9
11.83
44.4
0.77
Flour, unfortified
12(2)
59.8
13.5
22.67
37.9
1.31
Material
sR
RSDR, %
R
HORRAT
Recovery, %
Single Grubbs (Lab ID) 15
2, 4
Bread, enriched
12(2)
114.3
12.9
11.31
36.2
0.72
2, 10
Pasta, enriched macaroni
13(1)
223.5
28.8
12.88
80.6
0.91
2
Cereal oats
14(0)
666.8
144.1
21.61
403.5
1.80
Cereal wheat
12(2)
1365.6
101.4
7.42
283.8
0.69
Cereal rice
14(0)
684.9
147.3
21.51
412.6
1.80
Tortilla
13(0)
34.8
18.4
52.94
51.5
2.82
Baking mix
14(0)
173.3
37.4
21.59
104.8
1.47
Standard Reference Material
12(0)
145.6
21.4
14.71
60.0
1.07
a
Double Grubbs (Lab ID)
4, 7
113
Laboratories retained after outliers removed (number of outlier laboratories removed in parentheses). Each laboratory ran one test per material.
(pteroylglutamic acid) is extracted from the matrix by a triple enzyme system. A protease and an amylase are used to digest the food matrix and aid in the release of folates. Desiccated chicken pancreas conjugase is used to hydrolyze folylpolyglutamates to folyldiglutamates which, along with folic acid, can be used by the assay organism. The freed folates are extracted and diluted with basal medium containing all required growth nutrients except folate, and the turbidity of the Lactobacillus casei subspecies rhamnosus growth response for the test samples is compared quantitatively to that of known standard solutions. The method allows the optional use of 96 well microtiter plates and a microplate reader in place of the standard test tube system for semiautomated turbidity determination. B. Apparatus and Materials (a) Spectrophotometer.—To read 20 ´ 150 mm (or 18 ´ 150 mm) test tubes (Agilent, Palo Alto, CA). (b) Water bath.—Covered, 37° ± 0.2°C, with rotary shaker. (c) Test tubes.—Disposable, glass, Borosilicate, 20 ´ 150 or 18 ´ 150 mm. (d) pH meter.—With long combination electrode. (e) Vortex mixer. (f) Volumetric flasks.—Class A, low actinic and clear; 25, 50, 100, 250, 500, and 1000 mL. (g) Repetitive pipetters.—50 mL capacity with 1.25 mL disposable tips; and adjustable digital 100–1000 mL. (h) Pipeting machine(s).—To deliver 1 and 5 mL aliquots. Optional pipetter.—12 channel to use with 96 well microtiter plates. (i) Volumetric pipets.—Class A; 1, 2, 3, 4, 5, 10, 20, 25, and 50 mL. (j) Centrifuge tubes.—Oak Ridge type 28.6 ´ 106.1 mm, polypropylene, reusable; and disposable, 50 mL.
(k) Test tube rack holder.—Large, with cover. To hold 4 or more test tube racks. (l) Filter paper.—No. 2V, 12.5 cm. (m) Balances.—Analytical, readability to at least 4 places; and top loading, 3 places. (n) Hot plate stirrer. (o) Syringe.—Fitted with long needles, sterilizable, capable of delivering 50 mL. (p) Desiccator. (q) Optional for tube assay.—Automated assay tube reading; Gilson (Middleton, WI) escargot fractionator Model SC-30 or Model 222 sample changer modified with air agitation system and connected to spectrophotometer with flow cell and either printer or computer, or Autoturb II diluter and reader (Mitchum-Schaefer Inc., Indianapolis, IN). (r) Optional microtiter plate system.—96 Well microtiter plate reader and 96 well microtiter plates. A reader with appropriate filter(s) for 570–630 nm and efficient software for calculation is suitable. Note: For some plate reader systems, blackwall microtiter plates may be necessary to prevent deviations in readings between edge row wells and center row wells. Use of microtiter plate systems requires filter-sterilization of solutions using 0.22 mm sterilization filter units (15 and 500 mL, and 1 L), available as Nalge Disposable Filterware (Nalge Nunc International, Rochester, NY) or Corning Disposable Filterware (Corning, NY). C. Reagents Use distilled or double-distilled water throughout. (a) Adenine sulfate. (b) Agar.—Bacto, Difco No. 0140-01 (BD, Franklin Lakes, NJ). (c) Agar.—Bacto Lactobacilli agar (Difco No. 0900-15).
DEVRIES ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 88, NO. 1, 2005 9
(d) Alpha-amylase.—From Aspergillus oryzae, 150–250 U/mg protein (burette), Sigma Chemical Co. (St. Louis, MO) No. A-6211 is suitable. (e) p-Aminobenzoic acid (PABA). (f) Antifoam.—Mix 1.5 mL Dow Corning (Midland, MI) Antifoam AF or B with 100 mL water, and mix. Shake before using. (g) Ascorbic acid. (h) L-Asparagine monohydrate. (i) Biotin. (j) Calcium pantothenate. (k) Casein hydrolysate.—Vitamin-free acid hydrolyzed (Humko Sheffield Chemical, Lyndhurst, NJ). Hy-Case amino, ICN Cat. No. 104864, is suitable. (l) Celite.—Optional as filter aid. (m) Conjugase source.—Chicken pancreas, desiccated, Difco Bacto Cat. No. 0459-12. No substitutions. (n) L-Cysteine×HCl. (o) Ferric sulfate heptahydrate. (p) Folic acid.—Reference standard USP Cat. No. 28600. (q) Glutathione. (r) Guanine hydrochloride. (s) Liver.—Difco No. 0133-01. (t) Niacin. (u) Octanol. (v) Protease.—From Streptomyces griseus, powder Type XIV ca 4 U/mg solid, Pronase E from Sigma Chemical Co. No. P-5147. (w) Pyridoxine hydrochloride. (x) Riboflavin. (y) Sodium ascorbate. (z) Thiamin hydrochloride. (aa) D,L-Tryptophan. (bb) Uracil. (cc) Xanthine. (dd) Acetic acid.—0.02M. Dissolve 1.2 mL glacial acetic acid to ca 500 mL water. Dilute to 1 L with water. (ee) Ammonium hydroxide (NH4OH; 2 + 3).—Carefully mix 2 volumes concentrated ammonium hydroxide with 3 volumes water. (ff) Hydrochloric acid (HCl; 1 + 1).—Carefully mix equal volumes concentrated HCl and water (see Caution). (gg) Phosphate buffer (extract buffer).—pH 7.8. Dissolve 1.42 g sodium phosphate dibasic and 1.0 g ascorbic acid in water and dilute to 100 mL. Adjust pH to 7.8 with 4M NaOH. Prepare fresh on day used. Assay requires ca 35 mL buffer for each test sample and standard. (hh) Phosphate buffer (assay buffer).—pH 6.8. Dissolve 1.42 g sodium phosphate dibasic and 1.0 g ascorbic acid in ca 85 mL water and dilute to 100 mL. Adjust pH to 6.8 ± 0.02 with 4M NaOH. Prepare fresh on day used. Assay requires 12–15 mL buffer for each test sample and standard tube for test tube assay; ca 2.5 mL buffer for each test sample and standard for microtiter plate assay option. (ii) Phosphate buffer (for making standard solutions).—0.1M, pH 7.0. Dissolve 13.61 g potassium
phosphate monobasic in water and dilute to 1 L with water. Adjust pH to 7.0 with 4M potassium hydroxide. (jj) Potassium hydroxide.—4M. Dissolve 224 g potassium hydroxide in ca 500 mL water. Caution: Cool; dilute to 1 L with water. (kk) Sodium hydroxide.—4M. Dissolve 160 g sodium hydroxide in ca 500 mL water. Caution: Cool; dilute to 1 L with water. (ll) Sodium hydroxide.—0.01M. Pipet 2.5 mL 4M sodium hydroxide into 1 L volumetric flask. Dilute to volume with water. (mm) Adenine–guanine–uracil solution.—Dissolve 1.0 g each of adenine sulfate, C(a), guanine hydrochloride, C(r), and uracil, C(bb), in 50 mL warm HCl (1 + 1), C(ff), cool, and dilute with water to 1 L. Store at 10°C. Prepare fresh every 3 months. (nn) Xanthine solution.—Suspend 1.0 g xanthine, C(cc), in 150–200 mL water, heat to ca 70°C, add 30 mL NH4OH (2 + 3), C(ee), and stir until solid dissolves. Cool and dilute to 1 L with water. Store at 10°C. Prepare fresh every 3 months. (oo) Asparagine solution.—Dissolve 10 g L-asparagine monohydrate, C(h), in water and dilute to 1 L. Store at 10°C. (pp) Vitamin solution for folate.—Dissolve 10 mg PABA, C(e), 40 mg pyridoxine hydrochloride, C(w), 4 mg thiamin hydrochloride, C(z), 8 mg calcium pantothenate, C(j), 8 mg niacin, C(t), and 0.2 mg biotin, C(i), in ca 300 mL water. Add 10 mg riboflavin, C(x), dissolved in ca 200 mL 0.02M acetic acid, C(dd). Add solution containing 1.9 g anhydrous sodium acetate and 1.6 mL acetic acid in ca 40 mL water. Dilute to 2 L with water. Prepare fresh every 2 months. (Not necessary to prepare if using commercial basal medium preparation in reagent, C(uu)). (qq) Saline.—Sterile. Dissolve 9 g NaCl in 1 L water. Dispense 10 mL portions to 20 ´ 150 mm test tubes capped with plastic top. Sterilize 15 min at 121°–123°C and store in refrigerator. Prepare fresh weekly. (rr) Mixed salts solution.—Dissolve 20 g MgSO4×7H2O, 1 g NaCl, 1 g FeSO4×7H2O, and 1 g MnSO4×H2O in water. Dilute to 1 L. Add 1 mL concentrated HCl. Prepare fresh every 3 months. (ss) PABA–vitamin B6 solution.—Dissolve 50 mg PABA, C(e), and 120 mg pyridoxine hydrochloride, C(w), in 200 mL water. Add 0.95 g sodium acetate and 0.8 mL acetic acid to ca 40 mL water. Combine the 2 solutions and dilute to 500 mL with water. Store at 10°C. (tt) Agar maintenance medium.—Into 1 L hot water containing 10 mL 100 ng/mL vitamin B12, dissolve 48 g Lactobacilli agar, C(c), and 3 g Bacto agar, C(b). After agars dissolve, dispense 10 mL portions to 20 ´ 150 mm test tubes, and cap. Cover tubes to prevent contamination, autoclave 15 min at 121°–123°C, and store in refrigerator. Will keep indefinitely. (uu) Folic acid-free, double strength basal medium.—Prepare as in Table 2004.05B. [Alternatively, commercially available Difco Folic Acid Casei Medium (No. 0822-15) can be used. Prepare as directed: Suspend 9.4 g Difco Casei Medium and 50 mg ascorbic acid in 100 mL water, boil
10 DEVRIES ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 88, NO. 1, 2005 Table 2004.05B. Preparation of basal medium Amount of basal medium to prepare, mL 250
500
1000
5
10
Add reagents in order listed, mL Adenine–guanine–uracil solution, C(mm)
2.5
Xanthine solution, C(nn)
5
10
20
Asparagine solution, C(oo)
15
30
60
Vitamin solution for folate, C(pp)
50
100
200
5
10
20
5
10
5
10
20
40
Mixed salts solution, C(rr) PABA–vitamin B6 solution, C(ss)
2.5 Add ca 100 mL water and the following solids, g
Vitamin-free casein, hydrolyzed
2.5
Dextrose
10
Potassium phosphate, dibasic
0.25
0.5
1
Potassium phosphate, monobasic
0.25
0.5
1
33.2
66.4
Sodium acetate–3H2O
16.6
Glutathione, C(q)
0.00125
0.0025
0.005
0.125
0.25
0.5
0.05
0.1
0.2
Dissolve the following solids, g, in dilute HCl and add to above solution L-Cysteine×HCl, C(n) D,L-Tryptophan, C(aa)
Mix well, adjust to pH 6.8 with NaOH, and dilute to 1 L with water
for 1–2 min, and cool.] Dispense 200 mL portions into 16 oz polyethylene bottles, cap, and freeze (use within 2 months). (vv) Standard solutions.—(Use low actinic glassware.) (1) Stock solution.—100 mg/mL. Accurately weigh 50 mg USP folic acid, C(p), that has been dried to constant weight in vacuo at 70°C and stored in dark over P2O5 in desiccator and dissolve in 0.1M, pH 7.0, phosphate buffer, C(ii), in 500 mL volumetric flask. Dilute to volume with 0.1M phosphate buffer. Top with enough toluene to keep surface covered (usually 3–5 mL). Store in refrigerator. Check purity of standard and verify concentration of stock solution by pipetting 10 mL stock solution to 100 mL volumetric flask and diluting to volume with 0.1M, pH 7.0, phosphate buffer. Measure absorbance of solution at 282 and 346 nm in AUs using 0.1M, pH 7, phosphate buffer as a blank. Folic acid concentration in the stock solution: Folic acid, mg/mL = absorbance/absorptivity ´ dilution factor ´ 1000 ´ MW where absorptivity = 27 600 at 282 nm and 7200 at 346 nm, MW = 441.4. (2) Working standard solution.—1 mg/mL. Dilute 5 mL stock solution to ca 475 mL with water and adjust pH to ca 7.5 with HCl. Dilute to 500 mL with water. Prepare fresh on day of use. This will be diluted further when standards are run in parallel with samples.
(3) Diluted standard solution.—100 ng/mL. For use in working inoculum. Dilute 10 mL working standard solution, C(vv)(2), to ca 90 mL with water and adjust pH to ca 7.5 with HCl. Dilute to 100 mL with water. Top with enough toluene to keep surface covered (usually 3–5 mL). Store in refrigerator. Prepare fresh on day of use. (ww) Liquid culture medium.—50% Basal medium with 0.4 ng/mL folic acid and 10 mg/mL solubilized liver. Suspend 0.1 g liver, C(s), in 100 mL water. Hold mixture for 1 h at 50°C, and filter. Top with enough toluene to keep surface covered (usually 3–5 mL). Store in refrigerator. Pipet 20 mL to 1 L volumetric flask. Add (via pipet) 8 mL diluted folic stock solution (100 ng/mL), C(vv)(3). Dilute to volume with water. Mix equal volumes of solution with basal medium solution, C(uu). Dispense 10 mL portions diluted medium to 20 ´ 150 mm screw-cap test tubes, autoclave 15 min at 121°–123°C, and cool tubes rapidly. Store in refrigerator. Difco Bacto micro inoculum broth No. 0320-02 is also satisfactory as liquid culture medium. Will keep indefinitely. (xx) Conjugase solution.—Chicken pancreas solution (5 mg/mL). Weigh 0.5 g chicken pancreas, C(m), and add 100 mL buffer, pH 7.8, C(gg). Stir vigorously for 10 min. Transfer to 20 ´ 150 mm test tubes and centrifuge for 10 min at 2000 rpm. Decant supernatant through glass wool pledget into beaker, cover with parafilm, and store in refrigerator. Prepare fresh on day of use. Each test solution and standard requires 4–5 mL conjugase solution.
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(yy) Alpha-amylase solution.—20 mg/mL. Dissolve 0.5 g alpha-amylase, C(d), in 25 mL water. Store in refrigerator. Prepare fresh on day of use. Each test solution and standard requires 1 mL alpha-amylase solution. (zz) Protease solution.—2 mg/mL. Dissolve 0.05 g protease, C(v), in 25 mL water. Filter through glass wool pledget if necessary and store in refrigerator. Prepare fresh on day of use. Each test solution and standard requires 1 mL protease solution. D. Inoculum (a) Test organism.—Lyophilized L. casei subspecies rhamnosis, ATCC 7469. (b) Cryprotected inoculum.—Dissolve 4.7 g Difco Folic Acid Casei Medium in 50 mL distilled water. Heat to boiling, cool in ice, then add 50 mL water and 25 mg sodium ascorbate. Add 0.5 mL diluted folic standard solution, C(vv)(3), mix, and sterile-filter. Suspend one vial lyophilized L. casei in 1.0 mL of above medium using sterile techniques. Transfer 0.15 mL of this suspension to culture medium. Incubate at 37°C for 18 h. Mix 120 mL glycerol and 30 mL water, autoclave, and cool in ice bath. Cool incubated bacterial culture in ice bath and add 100 mL of the cold 80% glycerol. Mix gently but well. Aliquot 2.0 mL aliquots into sterile tubes (stir suspension occasionally to maintain even suspension). Can be stored at –20°C for up to 3 months and at –70°C for up to 6 months. (c) Working inoculum.—For tube cultures, dilute 2.0 mL frozen suspension, (b), to 50 mL with sterile saline. Mix on a Vortex mixer. Use this suspension as working inoculum. For 96 well plates, add 5 mL sterile saline to 2.0 mL solution direct from freezer. Mix on a Vortex mixer. Use this suspension as working inoculum. E. Preparation of Test Solutions (a) Product preparation.—Grind solids to pass 40 mesh sieve and store in air-tight containers, free from light exposure. (b) Hydrolysis and extraction.—Use distilled water unless otherwise indicated. (1) Standard.—10 ng/mL. Pipet 1.0 mL working standard solution (1 mg/mL), C(vv)(2), to 125 mL Erlenmeyer flask containing 20 mL phosphate buffer, pH 7.8, C(gg), mix, and add 30 mL water. (2) Test samples.—Accurately weigh test portion equal to 0.25–1.0 g dry basis solids containing ca 1 mg folic acid into respective 125 mL Erlenmeyer flask. Add 20 mL phosphate buffer, pH 7.8, and mix thoroughly. (If product is low in folate, do not take >1.0 g. To compensate for lower levels, use a larger aliquot in the second serial dilution below.) Dilute with water to 50 mL. Add 0.1–1.0 mL octanol (antifoam) to all flasks. Cover flasks with 50 mL beaker, autoclave 15 min at 121°–123°C, and cool. Add additional 10 mL buffer to each flask. Add 1 mL protease solution and incubate 3 h at 37°C. Inactivate enzyme by autoclaving (or placing in boiling water bath) 3 min at 100°C, and cool. Add 1 mL alpha-amylase solution to each flask, cover, and incubate 2 h at 37°C. Add 4 mL conjugase solution, cover, and incubate 16 h at 37°C. Inactivate enzymes by autoclaving 3 min at 100°C, and cool. Adjust tests and
standards to pH 4.5 with HCl (1 + 1) and dilute to 100 mL with water. Filter ca 20 mL through 2V filter paper. Use of Celite is acceptable to obtain clear filtrate. (Note: Filtered solutions can be set aside in the dark at 4°C overnight.) (3) Options.—For standard tube/spectrometer assay, proceed to next step, E(c). If using 96 well microtiter plate, filter-sterilize ca 5 mL filtered solution, using 0.22 mm filter system, into sterile screw-cap vials and store in dark at 4°C until ready to assay. Proceed directly to F(b). (c) Diluted standard and sample extracts (test tube method only).—(1) Standard.—0.3 ng/mL. Pipet 15 mL standard extract into 500 mL volumetric flask. (2) Test samples.—Pipet 10 mL of each test sample extract into respective 500 mL volumetric flasks. Add equal volume phosphate buffer, pH 6.8, to each standard and test flask. (If additional serial dilutions are necessary, add equal volume of buffer to volume pipetted to flask.) Dilute to volume with water. If concentration of folate is low, use larger aliquot; if too high, use smaller aliquot or larger volumetric flask. F. Determination (a) Assay using test tubes.––(Use of Brewer automatic pipetting machine is recommended.) Prepare twenty-one 20 ´ 150 mm tubes to contain 0, 0, 0.02, 0.04, 0.06, 0.08, and 0.10 ng/mL folic acid by adding in triplicate 0, 0, 1, 2, 3, 4, and 5 mL diluted (0.3 ng/mL) standard extract, E(c), into the tubes. Three 0 mL tubes will be used as uninoculated blanks. Prepare twelve 20 ´ 150 mm tubes for each test sample solution. In triplicate, place 1, 2, 3, and 4 mL diluted test sample extracts into the tubes. Add water to each to a total volume of 5 mL. Add 5.0 mL folic acid-free double strength basal medium, C(uu), to each tube. Cover tubes to prevent bacterial contamination. Autoclave 6 min at 121°–123°C. Cool tubes as rapidly as possible to
