Macroanalytical Procedures Manual (MPM) Main Page

Contents

1. Method for Apple Products (V-42)
2. Method for Decomposition in Blackberries, Raspberries, and Other Drupelet Berries (V-44)
3. Method for Decomposition in Blueberries (V-46)
4. Method for Cberries (V-48)

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A. Method for Apple Products (V-42)

(1) Scope

This method describes procedures for:

• Determination of rot fragments in apple butter as a supplement to the mold count method, AOAC 975.51. The rot fragment method permits examination of a large sample, which may provide additional information as to the type of decomposition present.
• Determination of extraneous filth and mold in unfiltered apple juice and vinegar stock.

Dried apple slices or chops are covered in Section 9.F., Dried Fruits.

Generally, in most pulped or extracted apple products, defective material is reduced to microscopic size, which requires analysis by digestive flotation separation procedures and by mold mycelia count techniques described in AOAC.

Most apple varieties are derived from Malus sylvestris Mill., of European and Western Asian origin, but some have originated through hybridization with other species of Malus. Apple butter is made by boiling down apples with sugar and spices. Apple pomace (cider press cake) is the solid residue remaining when raw apples are crushed and 80-90% of the fruit is pressed or extracted into juice. Apple pomace is used as a source for the manufacture of pectin; or the dried pomace residue can be fed to livestock. Nearly all known apple varieties have been used for apple juice. In order to obtain the desired flavor in the finished product, the common practice is to use a blend of two or more varieties.

(2) Applicable Documents

1. CPG 7110.01 Defect Action Levels for Apple Butter

(3) Defects

1. Insect Infestation and Damage -- Apple products may be prepared from rotten or worm-damaged fruit or from fruit contaminated in storage by insects. Rosy apple aphids [Dysaphis plantaginea (Passerini)] cause stunted, malformed fruit. Larvae of the codling moth [Cydea pomonella (L.)] and the apple maggot [Rhagoletis pomonella (Walsh)] make tunnels through the fruit. Apple curculio [Tachypterellus quadrigibbus (Say)] and plum curculio [Conotrachelus nenuphar (Herbst)] cause egg punctures and larval and adult feeding punctures, leading to the production of knotty, malformed fruit. Seasonal build-ups of large numbers of Drosophila flies may occur and result in contamination of products with eggs, maggots, and adults if effective measures of control are not taken.
2. Fungal Deterioration and Moldiness -- Deficiencies in handling and storage may promote the development of rot-producing fungi. A small amount of rot may produce a musty taste in a large amount of apple juice. Blue mold rot, caused by Penicillium expansum, is the most common and destructive. Brown rot, caused by Monilinia fructicola (Wint.) Honey, is found in all producing areas in the Eastern United States. Neofabraea perennans and N. malicorticis cause bull's-eye rot, especially in the Pacific Northwest. Black rot, caused by Physalospora obtusa, is common east of the Rocky Mountains. Mycosphaerella pomi, which causes fruit spot, is most destructive in New England, appearing in orchards which have not been properly sprayed.

(4) Procedure: Determination of Decomposition in Apple Butter

1. Rot Fragment Count -- Weigh 1.0 g apple butter into a 50 mL beaker. Add 20 mL water and about 2 drops 10% crystal violet in alcohol. Stain for 3 min. Decant through No. 100 sieve, rinse contents of sieve with 100-200 mL water, and quantitatively transfer retained pulp to graduated cylinder. Transfer is most readily accomplished by rinsing pulp to one edge of sieve, scraping out most of the pulp with a spatula, and then rinsing the sieve with water from a pipette or wash bottle to transfer the remaining pulp. Do not use more than 10 mL water to transfer during this operation. Fill to 10 mL mark with water and then with stabilizer solution [III. (15)] to 20 mL mark and mix well. Pipette two separate 0.5 mL portions and spread evenly over two rot fragment counting plates, 945.75B(q), using pipette as in AOAC 945.75B(p). Examine each slide, using widefield microscope at 40-45X and transmitted light. Count number of rot fragments per slide, add results and multiply by 20 to obtain the number of rot fragments per g of product.
2.  Mold Count -- Use AOAC 975.51.

(5) Procedure: Determination of Decomposition by Mold Count of Apple Pomace

Weigh 200 g pomace in a tared 1500 mL beaker and add 1000 mL water. Heat to boiling and simmer 2 hr. Add sufficient water to bring mixture to the original volume. Pulp through laboratory cyclone [945.75B(g)] and mix thoroughly. Weigh 50 g stabilizer solution [III. (15)] in a 250 mL beaker and add 50 g of the pulp. Mix thoroughly and make mold count as directed in AOAC 965.41.

(6) Procedure: Determination of Extraneous Filth and Mold in Unfiltered Apple Juice and Vinegar Stock

1. Analysis for Insect and Other Extraneous Matter -- The unfiltered apple juice used for the manufacture of filtered apple juice, cider, and vinegar stock is hard to filter. For this reason, a flotation rather than a direct filtration is used. Maggots as well as flies and fly parts may be present. Measure the subsample (up to 1 L) and transfer to a 2 L trap flask. Extract as in AOAC 970.66(B)(b), using water and 35 mL heptane [Ill.(7)]. Examine papers microscopically at 10-30X. Carefully decant the material remaining in the trap flask, leaving about 100 mL. Filter on black bolting cloth and examine for maggots. Report the total number and size of each per liter of subsample.
2. b. Analysis for (Geotrichum) Mold -- Measure subsample up to 1 L and follow AOAC 974.34, reporting numbers of mycelial fragments per sample volume.

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B. Method for Decomposition in Blackberries, Raspberries, and Other Drupelet Berries (V-44)

(1) Scope

This method is limited to a macroscopic procedure for the determination of decomposition in drupelet berries, including fruits of this type known as bramble berries or caneberries. The method is applicable to frozen and canned whole berries and provides, as necessary, information relative to the extent of decomposition in the fruit to supplement that provided by the mold count method for measuring decomposition.

(2) Applicable Documents

1. CPG 7110.03 Defect Action Levels

(3) Defects

1. Moldiness and Fungal Deterioration -- Berries may become moldy while still in the field if they are not harvested soon after they are ripe. Prolonged rainy periods which may delay picking will further aggravate this condition. Molds can also develop in harvested fruit if it is held too long before processing. Insect damage also tends to promote the growth of molds.
2. Insect Infestation and Damage -- There are a number of insect pests that may invade the fruit. Raspberry fruitworms are beetle larvae which penetrate the flower buds and developing fruit. Common species are the western raspberry fruitworm (Byturus bakeri Barber) and the eastern raspberry fruitworm (B. rubi Barber). Redberry mite (Acalitus essigi (Hassan)), which is common in the Pacific Coast area, produces "redberry disease" by feeding around the base of the drupelets, causing the fruit to become brightly colored and hard. Drupelet berries are sometimes infested by field insects such as thrips, which occur in large numbers. Drosophila flies may be attracted to overripe and sour fruits. AOAC methods are available for the determination of insect contamination.

(4) Procedure: Determination of Decomposition in Drupelet Berries

1. Sample preparation -- Prepare canned and frozen berries for examination as follows:

   1. (i) Canned berries -- Each subsample should consist of a composite of two No. 2 cans or the approximate equivalent from the same code. Divide the subsample into two equal portions, including the juice, without damaging the berries. These portions will be used for the mold count and macroscopic examination, respectively.
   2. (ii) Frozen berries -- Each subsample (representing one barrel, 30-lb can or other large container) should consist of about 3 quarts of berries. Thaw and mix the 3 quarts to make one composite analytical unit. Mix thoroughly without damaging the berries. If the berries are in consumer size packages (12-16 oz.), thaw and thoroughly mix four such packages from the same code to make one composite analytical unit. Divide the composited unit, including juice, into two unequal portions: one of about two-thirds of the volume to be used for the mold count and one of about one-third of the volume to be used for visual examination.
2. Visual Examination - Drain the berries (for frozen berries, use the second, smaller portion of the analytical unit) on a No. 20 sieve and wash with water. Immerse berries in water in large white pan and keep all berries immersed during examination. Repeat washing if water is not fairly clear. Examine each berry under water in strong light. Pick out as rotten those berries and fragments which have at least 5 drupelets containing either external or internal mold or both. External mold may show hyphal masses extending from the fruit. Internal mold will discolor and affect the brightness of the individual drupelet. Confirm all questionable rot spots by examining a fragment of the tissue for mold under a compound microscope. Classify the tissue as rotten only when a substantial number of mold filaments are present (see Chapter IV, Special Techniques). Drain the good and the rotten berries separately for 2 min on a 5-in. No. 20 sieve. Weigh the good and the rotten berries.
3. Mold Count -- Follow AOAC 955.47
4. Report -- Tabulate results as follows:

   Code No. _______	Barrel or Sub. No.
   	1	2	3	etc.	Average
   Total wt of drained Berries (g)
   Drained wt of good berries (g)
   Drained wt of rotten berries (g)
   Percent of rotten berries
   Percent of mold count
   Remarks:

C. Method for Decomposition in Blueberries (V-46)

(1) Scope

This method is limited to a macroscopic procedure for the determination of decomposition in blueberries. The method is applicable to frozen and canned whole blueberries and provides, as necessary, information relative to the extent of decomposition in the fruit to supplement that provided by the mold count method for measuring decomposition.

(2) Applicable Documents

(3) Defects

1. Insect Infestation and Damage -- A number of insect pests may attack the fruit. The blueberry maggot [Rhagoletis mendax Curran], which is a variety of the apple maggot, lays its eggs in the ripe fruit, causing many berries to drop. Cherry fruitworm [Grapholita packardi Zeller] is found throughout the deciduous fruit growing areas of the United States. The eggs of this insect are laid on the fruits or stems. When hatched, the larvae bore into the blueberry and tunnel about until ready to pupate, when they leave through a large exit hole. AOAC methods are available for determination of insect contamination.
2. Moldiness and Fungal Deterioration -- Blueberries are subject to attack by many species of fungus both before and after harvest. Molds primarily affect wild berries rather than cultivated berries because growers have greater difficulty controlling natural sources of contamination in the wild berries.

(4) Procedure: Determination of Decomposition in Blueberries

1. Sample Preparation -- Prepare canned and frozen berries for examination as follows:

   1. (i) Canned blueberries -- Each subsample should consist of about 20 oz. or the contents of a No. 2 can. If the subsample is larger, mix thoroughly and remove 20 oz. Drain for 2 min on a No. 20 sieve. Weigh a 40 g representative aliquot of the berries into a small bottle or jar, saving the remaining (larger) portion of the subsample for the mold count. Cover the 40 g portion with bleaching solution (2 tsp. sodium potassium metabisulfite added to 500 mL water) and let stand until the blueberries are bleached. Drain and transfer to a white pan. Count total number of blueberries. Proceed as in (4)b.
   2. (ii) Frozen blueberries -- Thaw the frozen berries in original containers, as appropriate. In containers larger than a No. 2 can, remove a No. 2 canful or 20 oz. If smaller, examine each subsample separately. Do not drain but proceed as in (i) above, beginning "Weigh a 40 g. . .". Save the larger portion for the mold count.
2. Visual Examination -- Examine each blueberry under water, using a strong light. Separate the rotten from the good berries. Decayed tissue can be identified as it resists bleaching. Confirm all questionable rot spots by examination of a small piece of the tissue for mold, using the compound microscope. Classify the tissue as rotten only when a substantial number of mold filaments are present. Classify as rotten any blueberry in which the rot affects 1/4 or more of the surface area. Count the number of rotten berries found in each subsample.

3. Mold Count -- Make mold counts as follows:

   1. (i) Canned Blueberries -- Pulp the larger portion retained from (4)a.(i) through a laboratory cyclone [AOAC 945.75B(g)]. Mix the pulp thoroughly. Weigh out 50 g stabilizer solution [III. (15)] and add 50 g blueberry pulp. Mix thoroughly and make mold count as in AOAC 965.41. Before placing cover glass into position, check the pulp on the mold count slide under a widefield microscope and remove any seeds or seed fragments.
   2. (ii) Blueberries Frozen with No Added Sugar -- Pulp the larger portion retained from a.(ii) through a laboratory cyclone [AOAC 945.75B(g)]; dilute with a stabilizer and make mold count as in (4)c.(i) above.
   3. (iii) Blueberries Frozen with Sugar or Syrup -- Berries frozen with sugar or syrup may not yield a satisfactory pulp for mold counting without cooking. Boil the retained blueberries from (4)a.(ii) for 5 min or until tender. Pulp the sample while hot, dilute with stabilizer solution and make mold count as in (4)c.(i) above.
4. Report -- Tabulate results as follows:

   Code No. _____	Subsample No.
   	1	2	3	etc.	Average
   Total No. of Berries
   No. of Rotten Berries
   Percent Rotten Berries
   Percent Mold Count
   Remarks:

D. Method for Cherries (V-48)

(1) Scope

This method describes procedures for sample preparation and visual examination to determine the

• Percent of reject cherries present in brined or maraschino cherries as a result of maggot damage
• Percent of reject cherries present in fresh, canned or frozen cherries as a result of rot or insect damage.

(2) Applicable Documents

1. CPG 7110.04 Defect Action Levels
2. CPG 7110.12 Mold in Cherry Jam

(3) Defects

1. Insect Infestation and Damage -- Prior to picking, cherries may become infested with maggots of the cherry fruit fly or cherry maggot [Rhagoletis cingulata (Loew)] or the black cherry fruit fly [Rhagoletis fausta (Osten Sacken)]. These maggots live within the fruit and are released by mashing the cherries. They may then be recovered by sedimentation from fresh, canned, or frozen cherries. In brined and maraschino cherries the maggots are dehydrated and are not recovered by the maggot sedimentation methods. Brining makes the maggots almost transparent, and frequently the black mouth hooks are the first clue to their presence. Usually the maggot lives in the pit cavity and is removed during pitting, sometimes leaving the mouth hooks. Maggot tunnels in brined cherries are brown depressions in the fruit adjoining the pit cavity.

   Other insects which may damage the fruit include cherry fruitworm (Grapholita packardi Zeller), pandemis moth [Pandemis albaniana (Walker)], destructive pruneworm [Mineola scitulella (Hulst)], Syneta leaf beetle [Syneta albida (Le Conte)], cherry curculio [Tachypterellus consors cerasi (List)], plum gouger [Coccotorus scutellaris (LeConte)], and plum curculio [Conotrachelus nenuphar (Herbst)].

2. Moldiness and Fungal Deterioration -- Cherries may rot as a result of injuries received before or after harvesting or because of delays in processing. Since blemishes may sometimes resemble rot, microscopic examination to determine the presence of mold may be required. Cherries to be used in brining are generally harvested prior to being fully matured in color and texture. Thus, no method is needed for determination of rot in brined and maraschino cherries. Fungus diseases and their causative organisms include brown rot [Monilinia fructicola (Wint.) Honey], Rhizopus rot (Rhizopus nigricans), Cladosporium rot or green mold (Cladosporium sp.), blue mold rot (Penicillium sp.) gray mold rot (Botrytis sp.), and alternaria rot (Alternaria sp).

(4) Procedure: Determination of Reject Cherries Caused by Maggot Damage in Brined or Maraschino Cherries

1. Sample Preparation -- Remove a representative analytical unit of 100 cherries (200 if halves ) from each subsample.
2. Visual Examination -- Cut open each cherry and examine under good lighting with 10X magnification, as necessary to show extent of damage. Classify and count damaged cherries in accordance with the three reject categories listed in reporting table (c).
3. Report -- Tabulate results as follows:

   Code No. ______	Subsample No.
   	1	2	3	etc.	Average
   Total No. examined
   No. with Maggot(s) present
   No. with Maggot Mouth Hooks Present
   No. with Maggot Tunneling Covering 1/4 or More of Pit Area
   Total No. of Rejects
   Percent of Rejects
   Remarks:

(5) Procedure: Determination of Reject Cherries Caused by Rot (Mold) and/or Insects in Fresh, Canned, or Frozen Cherries

1. Sample Preparation -- Each subsample should consist of about 20 oz. (No. 2 or No. 303 can). Drain juice from the cherries if present; count and record the number of cherries in each subsample. Place cherries in a large, flat white pan and cover with water. Drain and re-cover if water is not fairly clear.

2. Classification of Reject Cherries -- Reject cherries should be classified as follows:

   1. (i) Moldy -- Any cherry showing visible mold (hyphae and/or fruiting bodies) or a rotten area 6 mm or more in diameter. Confirm all questionable rotten areas by examination of a portion of the suspect tissue for the presence of mold with the compound microscope.
   2. (ii) Insect-Damaged -- Any cherry containing one or more whole or equivalent insects.
3. Visual Examination -- Examine each cherry under good lighting for mold or insects visible to the naked eye, or with such magnification as necessary in any particular case. If the magnification exceeds 10X, this should be stated in the report of results. Cut open any cherry as necessary to expose damage by mold or insects. Classify and count cherries in each reject category.
4. Report -- Tabulate results as in (4)c., substituting the appropriate savect categories.